CN106511347A - Applications of nitidine chloride in preparing medicines for preventing/treating sepsis - Google Patents

Abstract

The invention provides applications of nitidine chloride in preparing medicines for preventing/treating sepsis. According to the pharmacodynamics study on the prevention and treatment for the sepsis by adopting nitidine chloride, the prevention and treatment effects of nitidine chloride on the sepsis are studied from the aspects of survival rate, histopathologic changes and serum inflammatory factors of LPS-induced sepsis mice. Nitidine chloride can obviously improve the survival rate of the sepsis mice to 80%, obviously improve the histopathologic changes of the liver and lung tissues of the sepsis mice, reduce the level of proinflammatory factors in the serum of the sepsis mice, and increase the level of anti-inflammatory factors. The cell adoptive transfer experiment proves that for the sepsis mice injected through caudal vein, the survival rate of the sepsis mice is obviously improved, and therefore, nitidine chloride can be used for preparing the medicines for preventing/treating the sepsis. The medicine provided by the invention is a medicinal composition composed of the active ingredient, namely, nitidine chloride and the pharmaceutic adjuvants, and belongs to a preparation for oral administration, injection or percutaneous drug delivery.

Description

Application of the nitidine chloride in preventing/treating medication for treating pyemia is prepared

Technical field

The present invention relates to Chinese medicine, and in particular to application of the Chinese medicine in medicine is prepared, more particularly to application of the Chinese medicine monomer nitidine chloride in preventing/treating medication for treating pyemia is prepared.

Background technology

Radix Zanthoxyli, also known as double dorsal stylets, Radix Zanthoxyli etc., it is the dry root of Rutaceae xanthoxylum Radix Zanthoxyli [Zanthoxylum nitidum (Roxb.) DC], main product is in the Guangxi of China, Guangdong, Yunnan and other places.The effects such as with wind-dispelling removing dampness, reducing swelling and alleviating pain, be conventional Chinese medicine among the people, is used for treating the various disease conditions such as toothache, laryngopharynx swelling and pain, venom.Clinically also it is widely used, the medicine developed based on Radix Zanthoxyli has rheumatism liquid, reducing swelling and alleviating pain essence, Radix Zanthoxyli injection etc., is used for the diseases such as orthopaedics pain, toothache, gynecological inflammation, scytitiss.Modern pharmacological research shows that Radix Zanthoxyli has the various actives such as analgesia, antiinflammatory, antibacterial and antitumor.

Nitidine chloride (Nitidine Chloride) is to extract a kind of benzene phenanthridine alkaloid for obtaining from the dry root of Radix Zanthoxyli plant, and chemical structural formula is as follows：

Nitidine chloride is the main pharmacodynamics composition of Radix Zanthoxyli, with the extensive pharmacologically active such as antibacterial, malaria, antiinflammatory and antitumor.It is verified that nitidine chloride can significantly inhibit the activity of typeⅠtopoisomerase, it is potential antitumor drug.But it is less for the antiinflammatory action report of nitidine chloride, only document report nitidine chloride can be by adjusting MAPK and NF- κ B paths at present, the release and expression of proinflammatory inflammation factor in the RAW264.7 macrophages that suppression LPS (lipopolysaccharide) is activated.(Bouquet J,Rivaud M,Chevalley S,et al.Biological activities of nitidine,a potential anti-malarial lead compound[J].Malaria Journal,2012,11:67.)

Sepsis are the serious inflammatory reactions caused by infection, the damage of release and multiple organ along with a large amount of inflammatory mediators.But report is had no currently for the pyemic effect of nitidine chloride preventing and treating.Nitidine chloride can suppress the secretion of the proinflammatory inflammation factor of M1 type marks, promote the secretion of RM mark IL-10 (interleukin 10), then whether nitidine chloride has the function of adjusting macrophage polarization？For these problems, the present inventor have studied preventive and therapeutic effect of the nitidine chloride to mouse sepsis in whole animal level；And then the adjustment effect that nitidine chloride polarizes to macrophage is have studied using two kinds of Macrophage Models in vitro；Nitidine chloride be have studied on here basis and prevent and treat pyemic Pharmacodynamical mechanism, impact i.e. in vivo to macrophage polarization, further confirm nitidine chloride and the pyemic effect of preventing and treating is played by adjusting macrophage polarization, need further to develop its drug effect.

The content of the invention

The technical problem to be solved is that research nitidine chloride prevents and treats pyemic drug effect, further confirm nitidine chloride and the pyemic effect of preventing and treating is played by adjusting macrophage polarization, design nitidine chloride and apply in the new medicine for adapting to for the treatment of is prepared.

The invention provides application of the nitidine chloride in preventing/treating medication for treating pyemia is prepared.

Jing serial experiments of the present invention confirm nitidine chloride by adjusting macrophage polarization, so as to reach the pyemic effect of preventing and treating.

The present invention has carried out nitidine chloride and has prevented and treated pyemic pharmacodynamic study, have studied nitidine chloride to pyemic preventive and therapeutic effect by the sepsis mice induced to LPS in terms of survival rate, tissue pathologies change and serum levels of inflammatory cytokines three.Experimental result shows：Nitidine chloride can significantly improve the survival rate of sepsis mice and reach 80%.Nitidine chloride can be obviously improved the pathological change of sepsis Mouse Liver, lung tissue.Nitidine chloride can substantially reduce the level of proinflammatory inflammation factor in sepsis mice serum, raise the level of anti -inflammatory cytokine.Impact experiment of the nitidine chloride to RAW264.7 macrophage proliferations and apoptosis have studied using mtt assay and FCM methods, as a result show that 1-4 μM of nitidine chloride is not affected on the propagation of RAW264.7 macrophages and apoptosis, it is 0-4 μM that nitidine chloride acts on the safe dose of RAW264.7 macrophages.Impact experiment of the nitidine chloride to RAW264.7 macrophages secrete IL-10, because IL-10 is the most typical mark of modulability macrophage, and itself has critically important biological function, has very strong inhibitory action to inflammation.Test result indicate that nitidine chloride can promote the RAW264.7 macrophages secrete IL-10 of LPS activation with dose-dependant and time dependent mode.Further in the regulating and controlling effect experiment that nitidine chloride polarizes to macrophage, the impact experiment by nitidine chloride to RAW264.7 macrophages secrete IL-10, as a result nitidine chloride is not affected on the propagation of RAW264.7 macrophages；Impact experiment of the nitidine chloride to RAW264.7 macrophage apoptosis, shows that 1-4 μM of nitidine chloride can not cause the apoptosis of RAW264.7 cells.Nitidine chloride can promote the secretion of modulability macrophage marker IL-10, it was demonstrated that nitidine chloride can promote the conversion of the macrophage of classical activation to modulability macrophage.Nitidine chloride can promote the expression of modulability macrophage marker IL-10, SPHK1, LIGHT in the classical activated macrophage mark IL-12p40 of the different degrees of suppression of different LPS stimulation times points, the expression of TNF-α.The above results show that nitidine chloride can promote macrophage to convert from classical activation hypotype to modulability hypotype in vitro.For internal pharmacodynamic study, the present invention has carried out impact of the nitidine chloride to sepsis mouse spleen macrophage phenotype marker expression, as a result show that nitidine chloride can significantly suppress the expression of M1 marks in hepatic tissue, promote the expression of RM marks.Nitidine chloride can significantly suppress the expression of M1 marks in lung tissue, promote the expression of RM marks.Prove that nitidine chloride can promote the conversion of sepsis Mouse Liver, macrophage in lung tissues from M1 to RM in vivo, nitidine chloride may be relevant with regulation and control macrophage phenotype polarization for the preventive and therapeutic effect of mouse sepsis.Nitidine chloride impact in vivo to macrophage polarization, as a result show that nitidine chloride can significantly promote the expression of modulability macrophage outstanding feature thing IL-10, SPHK1 and LIGHT in sepsis mouse spleen macrophage and liver, lung tissue, suppress classical activated form macrophage outstanding feature thing IL-12p40, TNF-α, the expression of IL-6 and iNOS, convert to modulability macrophage so as to show that nitidine chloride promotes classical activated form macrophage in vivo.Description of test nitidine chloride is relevant with its adjustment effect to macrophage polarization for pyemic preventive and therapeutic effect, the present invention is verified using cell adoptive transfer experiment, experimental result shows, the macrophage of In vitro culture derived from bone marrow, Jing after LPS is activated and nitidine chloride is processed, which is made to be polarized to modulability macrophage, Jing tail veins give sepsis mice, can significantly improve the survival rate of sepsis mice.Therefore, tested by the drug efficacy study of inside and outside, it was demonstrated that nitidine chloride can be used to prepare preventing/treating medication for treating pyemia.

Preventing/treating medication for treating pyemia of the present invention is the pharmaceutical composition being made up of as active component and pharmaceutic adjuvant nitidine chloride.

Pharmaceutical composition of the present invention is oral formulations, injection or percutaneous drug delivery preparation.

The present invention provides and confirms the pyemic preventive and therapeutic effect of nitidine chloride, can be further developed as medicine.The present invention provides new anti-medication for treating pyemia for clinic, has larger using value.

Description of the drawings

The survival rate wherein vertical coordinate rate for survival of the nitidine chloride observation sepsis mice of 1 lumbar injection various dose of Fig. 1 embodiments, abscissa are time point after injection LPS

△ represents lumbar injection 10mg/kg nitidine chlorides, and zero represents lumbar injection 3mg/kg nitidine chlorides, and represents lumbar injection 1mg/kg nitidine chlorides, when ◇ represents that the physiology salt waterline of the same volume of lumbar injection represents that t is checked, p value<0.05, when line represents that t is checked, p value<0.01, when line represents that t is checked, p value<0.001, show that the raising of the survival rate has significant difference；When the line not having represents that t is checked, p value>0.05, show that the raising of the survival rate does not have significant difference.

2 nitidine chloride of Fig. 2 embodiments is cut into slices to the up expression murine liver tissue of impact that sepsis Mouse Liver, pathologic state colony change, descending expression mouse lung tissue section, the left side first is classified as normal group mouse tissue, second is classified as model group mouse tissue, and the 3rd is classified as nitidine chloride administration group mouse tissue

2 nitidine chloride of Fig. 3 embodiments can substantially reduce the level of proinflammatory inflammation factor in sepsis mice serum, raise the level of anti -inflammatory cytokine.Abscissa represents various cytokines, and vertical coordinate represents each cytokine levels.Zero represents TNF-α level, and represents IL-6 levels, and △ represents IL-10 levels.

3 nitidine chloride of Fig. 4 embodiments does not have obvious inhibited proliferation to the macrophage of derived from bone marrow.Abscissa represents variable concentrations nitidine chloride, is from left to right followed successively by 0,1,2,4 μM of nitidine chlorides；Vertical coordinate is OD values.

3 nitidine chloride of Fig. 5 embodiments can not cause the apoptosis of the macrophage of bone marrow derived.

Abscissa is Annexin V-FITC antibody labelings, and vertical coordinate is PI antibody labelings.

The picture left above represents normal cell, and top right plot represents cell after 1 μM of nitidine chloride effect, and lower-left figure represents cell after 2 μM of nitidine chloride effects, and bottom-right graph represents cell after 4 μM of nitidine chloride effects

Fig. 6 embodiments 3 show that nitidine chloride can promote the secretion of RM mark IL-10 dose-dependant and time-dependent.

Left figure is impact secrete to IL-10 of various dose nitidine chloride, and vertical coordinate is IL-10 contents, and abscissa is from left to right followed successively by Control groups, LPS groups, μM nitidine chloride group of LPS+1,2,4.

Right figure is that different time adds impact secrete to IL-10 of nitidine chloride, and vertical coordinate is IL-10 contents, and abscissa is from left to right followed successively by Control groups, LPS groups, after LPS 1h, LPS simultaneously, 1h, 6h, 18h addition nitidine chloride group before LPS.When ### represents that t is checked, p value<0.001, show relative to Control groups, promotions of the LPS to cytokine secretion has significant difference；When line represents that t is checked, p value<0.05, when line represents that t is checked, p value<0.01, show that, relative to LPS groups, the raising of this group of IL-10 secretion has significant difference；When the line not having represents that t is checked, p value>0.05, show that the raising of this group of IL-10 secretion does not have significant difference.

Fig. 7 embodiments 3 show that nitidine chloride can suppress the secretion of M1 mark IL-12p70 and NO

Left figure is impact secrete to IL-12p70 of various dose nitidine chloride, and vertical coordinate is IL-12p70 contents, and abscissa is from left to right followed successively by Control groups, LPS groups, μM nitidine chloride group of LPS+1,2,4.Right figure is impact secrete to NO of various dose nitidine chloride, and vertical coordinate is NO contents, and abscissa is from left to right followed successively by Control groups, LPS groups, LPS+ positive drug groups, μM nitidine chloride group of LPS+1,2,4.When ### represents that t is checked, p value<0.001, show relative to Control groups, promotions of the LPS to cytokine secretion has significant difference；When line represents that t is checked, p value<0.05, when line represents that t is checked, p value<0.01, when line represents that t is checked, p value<0.001, show relative to LPS groups, the suppression of this group of cytokine secretion has significant difference.

Impact of 3 nitidine chloride of Fig. 8 embodiments to each phenotypic marker mRNA expression of macrophage of derived from bone marrow

Abscissa is different time points 0h, 1h, 4h, 16h after LPS stimulations, and vertical coordinate is each cytokine mRNA levels.It is up to be from left to right followed successively by RM mark IL-10, SPHK1, LIGHT, it is descending to be from left to right followed successively by M1 mark IL-12p40, TNF-α.

Impact of 4 nitidine chloride of Fig. 9 embodiments to sepsis mouse spleen macrophage phenotype marker expression

Abscissa is each phenotypic marker, and zero represents M1 type mark IL-12p40, TNF-α, IL-6, iNOS, and represents RM mark IL-10, SPHK1, LIGHT.Vertical coordinate is mRNA level in-site.

Impact of 4 nitidine chloride of Figure 10 embodiments to various macrophage phenotype mark mRNA expression in murine liver tissue

Abscissa is each phenotypic marker, and zero represents M1 type mark IL-12p40, TNF-α, IL-6, iNOS, and represents RM mark IL-10, SPHK1, LIGHT.Vertical coordinate is mRNA level in-site.

Impact of 4 nitidine chloride of Figure 11 embodiments to various macrophage phenotype mark mRNA expression in sepsis mouse lung tissue

Abscissa is each phenotypic marker, and zero represents M1 type mark IL-12p40, TNF-α, IL-6, iNOS, and represents RM mark IL-10, SPHK1, LIGHT.Vertical coordinate is mRNA level in-site.

Impact of the BMDMs adoptive transfers of 4 nitidine chloride of Figure 12 embodiments process to sepsis mouse survival rate

Vertical coordinate rate for survival, abscissa are time point after injection LPS

Zero represents direct lumbar injection 10mg/kg nitidine chlorides, and represents the macrophage of the derived from bone marrow that lumbar injection nitidine chloride is processed, when ◇ represents that the physiology salt waterline of the same volume of lumbar injection represents that t is checked, p value<0.05, when line represents that t is checked, p value<0.001, show that the raising of the survival rate has significant difference.

Specific embodiment

The impact of the survival rate of the sepsis mice that 1 nitidine chloride different dosing dosage of embodiment and different dosing time are induced to LPS.

1.1 experiment material

Nitidine chloride (commercially available, upper Hiroad standing grain company)；

(Sigma Co., USA, from escherichia coli 055 for LPS：B5)；

0.9% normal saline (laboratory self-control)；

1.2 laboratory animal

BALB/c mouse, male, 6-8 week old, 20-22g；There is provided by Shanghai Slac Experimental Animal Co., Ltd., raised in pharmaceutical college of The 2nd Army Medical College cleaning grade Animal House.In Animal House, keeping temperature is 21 ± 2 DEG C, 12 hours day-night cycle, and mice can ad lib drinking-water.Mice before the experiments were performed, is raised 7 days to adapt to environment.

1.3 experimental technique

1.3.1 the preparation of nitidine chloride sample

Weigh 10mg, 3mg, 1mg nitidine chloride powder, first plus 200 μ l DMSO dissolving, add 5ml normal saline, ultrasonic 20min makes which fully dissolve, finally the sample solution equivalent to mice 10mg/kg, 3mg/kg, 1mg/kg dosage is respectively obtained with normal saline constant volume to 10ml.

1.3.2 LPS induction mouse sepsis model foundation and dosage regimen

BALB/c mouse is randomly divided into model group and different dosing group, and per group of 10 mices, by LPS powder physiological saline solutions, every mouse peritoneal injection 15mg/kg LPS solution；

Dosage regimen：(1) investigate optimal dosage：Before modeling, 1h gives the nitidine chloride solution of mouse peritoneal injection 10mg/kg, 3mg/kg, 1mg/kg dosage respectively, and model group gives the normal saline of same volume.(2) Best administration time is investigated, 1h, lps injection are simultaneously after the lps injection, 1h, 6h, 18h give the nitidine chloride solution of mouse peritoneal injection optimal dose before lps injection, and model group gives the normal saline of same volume.

1.3.3 the observation of the sepsis mouse survival rate of LPS inductions

After the completion of the administration of each group mice, it is put back in the cage of each group, ad lib drinking-water, the survival rate change observed in 84h at interval of 12h, records dead mouse number, dead mice is taken out.

1.3.4 statistical analysis

The drafting of figure and the analysis of data are carried out using 5 softwares of Graphpad Prism according to a conventional method, the variation analyses to Kaplan-Meier survival curves are checked using log-rank, P<0.05 is considered to have statistical significance.

1.4 experimental result

Can engender after mice trans-abdominal chamber injection LPS shake, the symptom such as dyspnea, diarrhoea, activity then is reduced, is squeezed a ball, twitches, last dead.In the investigation of dosage, model group starts dead mouse phenomenon occur from 24h, and in dead 4 mices of 24h, now survival rate is 60%, and subsequent mice is gradually dead, and all dead in 60h, survival rate is 0；10mg/kg nitidine chloride administration group mices start dead mouse occur from 48h, and now survival rate is 80%, is gradually decreased to 50% to 60h survival rates, and 60h to 84h does not have dead mouse, and survival rate is maintained at 50%；3mg/kg nitidine chloride administration group mices start dead mouse occur from 36h, and now survival rate is gradually decreased to 40% for 80%, 60h survival rates, and 60h to 84h does not have dead mouse, and survival rate is maintained at 40%；1mg/kg nitidine chloride administration group mices start 2 dead mouses occur from 24h, and now survival rate is 80%, is down to 20% to 60h survival rates, and 60h to 84h does not have dead mouse, and survival rate is maintained at 20% (Fig. 1).As a result show that the nitidine chloride of various dose can improve the survival rate of sepsis mice, and the protective effect of nitidine chloride has dose dependent.

In the investigation of administration time, model group starts dead mouse phenomenon occur from 24h, and in dead 5 mices of 24h, now survival rate is 50%, and subsequent mice is gradually dead, and all dead to 60h, survival rate is 0；Before lps injection, 18h administration groups mice starts 1 dead mouse occur from 24h, and now survival rate is 90%, is gradually decreased to 60% to 60h survival rates, and 60h to 84h does not have dead mouse, and survival rate is maintained at 60%；Before LPS injections, 6h administration groups mice starts dead mouse occur from 48h, and now survival rate is down to 80% for 90%, 60h survival rates, and 60h to 84h does not have dead mouse, and survival rate is maintained at 80%；Before lps injection, 1h administration groups mice starts 3 dead mouses occur from 24h, and now survival rate is 70%, is down to 50% to 72h survival rates, and 72h to 84h does not have dead mouse, and survival rate is maintained at 50%；Lps injection is administered simultaneously group mice and starts 4 dead mouses occur from 24h, and now survival rate is 60%, is down to 10% to 72h survival rates, and 72h to 84h does not have dead mouse, and survival rate is maintained at 10%；After lps injection, 1h administration groups mice starts 5 dead mouses occur from 24h, and now survival rate is 50%, is down to 10% to 60h survival rates, and 60h to 84h does not have dead mouse, and survival rate is maintained at 10%.As a result show lps injection while and after lps injection, 1h administrations can not all significantly improve the survival rate of sepsis mice, and 1h, 6h, 18h are remarkably improved the survival rate of sepsis mice before lps injection, and most strong with the protective effect of 6h administrations before lps injection.

Brief summary：(1) in the nitidine chloride of same time lumbar injection various dose, by observing the survival rate of sepsis mice, it is determined that 10mg/kg is the optimal dosage of nitidine chloride.

(2) in the nitidine chloride of different time lumbar injection same dose, by observing the survival rate of sepsis mice, it is determined that before lps injection, 6h is the Best administration time of nitidine chloride.

The pyemic preventive and therapeutic effect research experiment that 2 nitidine chloride of embodiment is induced to LPS

2.1 experiment material

2.1.1 medicine and reagent

Nitidine chloride (upper Hiroad standing grain company)；

(Sigma Co., USA, from escherichia coli 055 for LPS：B5)；

0.9% normal saline (laboratory self-control)；

IL-10, TNF-α, the ELISA kit (eBioscience companies of the U.S.) of IL-6；

Paraformaldehyde (Shanghai Bo Guang companies)

2.1.2 instrument and equipment

Horizontal centrifuge Allegra X-30R Centrifuge (Beckman coulter, the U.S.)；

- 80 DEG C of ultra cold storage freezers (Thermo Fisher Scientific, the U.S.)；

Constant temperature blending instrument (Eppendorf Thermomixer comfort, the U.S.)；

Microplate reader microplate reader Elx800 (BioTek Instruments, the U.S.)；

2.2 laboratory animal

BALB/c mouse, male, 6-8 week old, 20-22g；There is provided by Shanghai Slac Experimental Animal Co., Ltd., raised in pharmaceutical college of The 2nd Army Medical College cleaning grade Animal House.In Animal House, keeping temperature is 21 ± 2 DEG C, 12 hours day-night cycle, and mice can ad lib drinking-water.Mice before the experiments were performed, is raised 7 days to adapt to environment.

2.3 experimental technique

2.3.1 the foundation and administration of the mouse sepsis model of LPS inductions

20 BALB/c mouse are randomly divided into model group and administration group, and per group of 10 mices, every mouse peritoneal injection 15mg/kg LPS solution carry out modeling；Administration group 6h before modeling gives the nitidine chloride solution of lumbar injection 10mg/kg, and model group gives the normal saline of same volume, for the observation of mouse survival rate.

30 BALB/c mouse are randomly divided into model group and administration group, per group of 15 mices, modeling and administering mode ibid, for determining mice serum inflammatory factor and observation liver, pathologic state colony change.

2.3.2 the observation of mouse survival rate

After the completion of each group mice modeling, it is put back in the cage of each group, ad lib drinking-water, the survival rate observed in 84h at interval of 12h, records dead mouse number, dead mice is taken out.

2.3.3 the change of Mouse Liver, pathologic state colony

16h after each group mice lps injection, lumbar injection 300mg/kg chloral hydrate anesthesias, open abdominal cavity, its liver, lung tissue are taken carefully, 24h is fixed in being put into rapidly the 50ml centrifuge tubes of the paraformaldehyde for filling 4% (notices that tissue will be sufficiently spread out, avoid crimp), with the ethanol dehydration of low concentration to high concentration, with transparent in dimethylbenzene, paraffin embedding, section, dewaxing dyeing (hematoxylin-eosin HE dyeing), basis of microscopic observation Mouse Liver, pathologic state colony change.

2.3.4 the change of ELISA method detection mice serum inflammatory factor

Each group mice 1h after lps injection plucks eyeball and takes blood, and after room temperature places 10min, horizontal centrifuge 12000rpm centrifugation 5min carefully take upper serum, and the serum of 5 mices of per group of each time point merges, be stored in -80 DEG C it is to be measured.The content of IL-10, TNF-α, IL-6 in serum is detected using eBioscience ELISA kits (enzyme-linked immunosorbent assay kit of eBioscience companies).

2.4 experimental result

2.4.1 the impact of the survival rate of the sepsis mice that nitidine chloride is induced to LPS

In experiment, model group starts dead mouse phenomenon occur from 24h, and in dead 3 mices of 24h, now survival rate is 70%, and subsequent mice is gradually dead, and all dead to 72h, survival rate is 0；Administration group 6h lumbar injections 10mg/kg nitidine chlorides before lps injection, start 2 dead mouses occur from 48h, and now survival rate is 80%, and 48h to 84h does not have dead mouse, and survival rate is maintained at 80%.As a result before showing modeling, 6h lumbar injections 10mg/kg nitidine chlorides can significantly improve the survival rate of the sepsis mice of LPS inductions.

2.4.2 the impact that nitidine chloride changes to sepsis Mouse Liver, pathologic state colony

In pyemic pathogenic process, endotoxin can cause internal a large amount of inflammatory cell infiltrations to multiple tissues such as liver, lung, the acute tissue injury of induction, it is further developed into the even exhaustion of multiple organ dysfunction sexual disorders, this is the dead major reason of sepsis subject, therefore this experiment takes normally (Control), model (Model) and administration (Nit) and organizes the liver of mice, lung tissue and carry out paraffin embedding, section and HE and dyes to observe the change in each group Mouse Liver, pathologic state colony to further determine that nitidine chloride to pyemic preventive and therapeutic effect.In the section of Control groups murine liver tissue, it is a central vein in the middle of lobules of liver, normally, hepatic cords are radial regularly arranged to surrounding centered on central vein for hepatocyte；In the section of Model groups murine liver tissue, hepatic cords irregular arrangement, hepatocyte are downright bad in a large number, structural fuzzy, and there is substantial amounts of inflammatory cell infiltration necrotic area；In the section of Nit groups murine liver tissue, hepatocyte is rare downright bad and no inflammatory cell infiltration phenomenon.In the section of Control groups mouse lung tissue, alveolar septum is normal, and alveolar wall thickness is moderate；Alveolar collapse in the section of Model groups mouse lung tissue, alveolar wall are seriously thickened；In the section of Nit groups mouse lung tissue, alveolar septum size tends to normal, and alveolar wall is rare to thicken (Fig. 2).As a result before illustrating lps injection, 6h lumbar injection 10mg/kg nitidine chlorides can effectively reduce Mouse Liver, the pathological change of lung tissue caused by sepsis.Impact of the nitidine chloride to sepsis mice serum inflammatory factor：Sepsis are a kind of systematic inflammatory reactions, early stage pathogenesis of sepsis, outburst causes serious damage and the increase of mortality rate to various proinflammatory inflammation factors in a large number, wherein the proinflammatory inflammation factor such as TNF-α, IL-6 is the critical mediator for causing body excessive inflammatory response, carries out suppression to which in morbidity early stage and may sepsis be played with good improvement result.IL-10 is a kind of crucial anti -inflammatory cytokine, and it can suppress the secretion of various proinflammatory mediators, and improves the survival rate of sepsis mice, plays very important effect in the preventing and treating of pathogenesis of sepsis early stage.Therefore this experiment have detected the change of model and administration group mice each inflammatory factor in 1h serum after lps injection using ELISA method.As a result find that nitidine chloride can significantly reduce the proinflammatory inflammation factor TNF-α in sepsis mice serum, the level of IL-6 and raise the level (Fig. 3) of anti -inflammatory cytokine IL-10.

Brief summary：(1) nitidine chloride can significantly improve the survival rate of sepsis mice and reach 80%.

(2) nitidine chloride can be obviously improved the pathological change of sepsis Mouse Liver, lung tissue.

(3) nitidine chloride can substantially reduce the level of proinflammatory inflammation factor in sepsis mice serum, raise the level of anti -inflammatory cytokine.

The impact that 3 nitidine chloride of embodiment polarizes to the macrophage of derived from bone marrow

Macrophage polarizes to M1 types macrophage under the stimulation of LPS, discharge a large amount of proinflammatory mediators and a small amount of anti-inflammatory medium, but macrophage is may result under the influence of some factors to convert from M1 types to RM, discharges the proinflammatory mediators of reduced levels and the anti-inflammatory medium of higher level.This experiment is separated and the macrophage for having cultivated bone marrow derived, the safe dose that nitidine chloride acts on the macrophage of derived from bone marrow is primarily looked at, and then mark secretion and the impact expressed of the nitidine chloride to M1 and RM is detected respectively, further to confirm whether nitidine chloride promotes conversions of the M1 to RM.

2.1 experiment material

2.1.1 primary cell

The macrophage (taking from mice before experiment) in Primary bone marrow source

2.1.2 material and reagent

70 μm of cells grinding filter screen (U.S. company BD)

DMEM complete mediums：

DMEM culture medium (Gibco companies of the U.S.)+10% hyclone (Gibco companies of the U.S.)+1% dual anti-(100units/ml penicillins, 100units/ml streptomycins are purchased from Sigma Co., USA)

1640 complete mediums：1640 culture medium (U.S. Gibco)+10% hyclone+1% is dual anti-

Recombined small-mouse cytokine M-CSF (Peprotech companies of the U.S.)

Biological level PBS (Hyclone companies of the U.S.)

IL-10, IL-12p70ELISA test kit (eBioscience companies of the U.S.)

FITC-Annexin V apoptosis detection kit (BD Pharmingen companies of the U.S.)

Thiazolyl blue (sigma companies of the U.S., MTT)

Total serum IgE extraction agent box (Chinese Fei Jie companies)

Reverse transcription reagent box (Japanese Takara companies)

Real-time fluorescence quantitative PCR test kit (Japanese Takara companies)

2.1.3 instrument and equipment

Constant temperature CO2 gas incubator (Japanese SANYO companies)；

Horizontal centrifuge Allegra X-30R Centrifuge (Beckman coulter companies of the U.S.)；

- 80 DEG C of ultra cold storage freezers (Thermo Fisher Scientific companies of the U.S.)；

Constant temperature blending instrument (U.S. Eppendorf Thermomixer comfort, USA)；

Enzyme mark detector (Biotec companies of the U.S.)；

Flow cytometer (FACS Calibur U.S. company BDs)；

Real-time fluorescence quantitative PCR instrument (American AB I company).

2.2 laboratory animal

C57BL/6 mices, female, 6-8 week old, 18-20g；There is provided by Shanghai Slac Experimental Animal Co., Ltd., raised in pharmaceutical college of The 2nd Army Medical College cleaning grade Animal House.In Animal House, keeping temperature is 21 ± 2 DEG C, 12 hours day-night cycle, and mice can ad lib drinking-water.Mice before the experiments were performed, is raised 7 days to adapt to environment.

2.3 experimental technique

2.3.1 the separation and culture of the macrophage of bone marrow derived

According to list of references, the macrophage for establishing bone marrow derived is separated and cultural method.

([1]Lili F,Pingping S,Hang Z.Pentamethoxyflavanone regulates macrophage polarization and ameliorates sepsis in mice[J].Biochemical Pharmacology,2014,89(1):109–118.[2]Qin H,Holdbrooks AT,et al.Benveniste,E.N.SOCS3deficiency promotes M1macrophage polarization and inflammation[J].The Journal of Immunology,2012,189(7):3439–3448.)

(1) make arrangements for surgery apparatus, sterilization；

(2) mice is put to death using the method for cervical dislocation, is placed in 75% ethanol, soak 2min.The skin of mice back leg is cut off with the shears for having sterilized in advance, both sides femur and tibia is carefully cut, is got rid of the muscular tissue on bone as far as possible；

(3) femur and tibia of acquirement are placed in the sterilized petri dishes for filling RPMI-1640 culture medium, RPMI-1640 culture medium is drawn with syringe, blow and beat repeatedly, medullary cell is blown out in insertion medullary cavity, until medullary cavity is white；

(4) blow and beat repeatedly cell and make single cell suspension, cell suspension is drawn by 70 μm of nylon cell strainers, collected in centrifuge tube；

(5), in 1500rpm centrifugation 5min, careful abandoning supernatant, (compound method is as follows for the erythrocyte cracked liquid of addition 10ml for the cell suspension collected：Weigh Tris 0.65g, NH₄Cl 1.87g, MilliQ water is settled to 250ml, and regulation PH is 7.2-7.4), uniform, stationary incubation 2min is blown and beaten repeatedly, the cell suspension for terminating is incubated and 5min is centrifuged in 1500rpm, careful abandoning supernatant, if now cell white then erythrocyte is abolished completely；

(6) the aseptic PBS cells of 10ml are added twice, 1500rpm centrifugation 5min, supernatant discarded, with 1ml RPMI-1640 culture medium re-suspended cells；

(7) cell counting, with the RPMI-1640 complete mediums containing 10ng/ml M-CSF, (collocation method is as follows：10 μ g recombined small-mouse cytokines M-CSF are dissolved in 100 μ l deionized waters, should not be vortexed, it is further dissolved in 900 μ l, 1640 complete mediums, every 50 μ l are sub-packed in 1.5ml centrifuge tubes, it is stored in -80 DEG C, take out before use and be added in 50ml culture medium, final concentration of 10ng/ml) with 2 × 10⁷The cell density dilution of/ml, is placed in 10cm plates, 5%CO₂, cultivate in 37 DEG C of incubators；

(8) experimental day is designated as the 0th day, old culture fluid was discarded in the 3rd day, aseptic PBS is washed twice, is added the fresh RPMI-1640 culture fluid containing 10ng/ml M-CSF, is every other day carried out afterwards changing liquid, discard culture supernatant within 7th day, 2ml trypsin digestion and cell 3min are added, adds culture medium of the 5ml containing serum to terminate digestion immediately, gently blown and beaten repeatedly plate and collect cell, bed board is counted, subsequent experimental is carried out.

2.3.2 the macrophage purity detecting of bone marrow derived

The macrophage of collection bone marrow derived is in 1.5ml centrifuge tubes, with the PBS of 4 DEG C of pre-coolings twice, it is resuspended in 500 μ l PBS, blank control group (not dyeing), PE-CD11b antibody or FITC-F4/80 antibody list dye group and double dye groups is divided into, each group cell density is not less than 1 × 10⁶/ ml, the PE-CD11b antibody and FITC-F4/80 antibody for being separately added into 5 μ l are dyeed, and after being incubated 30min, are detected with flow cytometer in the environment of being placed in room temperature lucifuge.

2.3.3 mtt assay detection impact of the nitidine chloride to the macrophage proliferation of derived from bone marrow

The macrophage of derived from bone marrow is collected, counted under microscope is diluted to 5 × 10 with DMEM complete mediums⁵The density of/ml, is seeded to 96 orifice plates, per 100 μ l of hole, 5%CO₂, cultivate 4h in 37 DEG C of incubators；After cell is fully adherent, add the nitidine chloride solution of variable concentrations (1 μM, 2 μM, 4 μM), per group of 3 multiple holes of setting, blank group (cell suspension) is set simultaneously, administration group (cell suspension+variable concentrations medicine), after effect 24h, tetrazolium bromide (MTT) solution of 10 μ l 5mg/ml is added per hole, continue incubation 4h；Careful suction abandons every hole supernatant, adds 100 μ l DMSO, and concussion mixes 10min, after purple crystal to be generated is completely dissolved, detects at microplate reader 570nm.

2.3.4 FCM methods detection impact of the nitidine chloride to the macrophage apoptosis of derived from bone marrow

The macrophage of derived from bone marrow is collected, counted under microscope is diluted to 5 × 10 with DMEM complete mediums⁵The density of/ml, is seeded to 6 orifice plates, per hole 2ml, 5%CO₂, cultivate 4h in 37 DEG C of incubators；After cell is fully adherent, add (1 μM of variable concentrations, 2 μM, 4 μM) nitidine chloride solution, blank group (cell suspension) is set simultaneously, administration group (cell suspension+variable concentrations medicine), after effect 24h, trypsinization collects cell, and (the trypsinization time is unsuitable long, infringement cell is avoided to cause false positive), with the PBS of 4 DEG C of pre-coolings once, 1500rpm is centrifuged 5min, add 500 μ l Binding Buffer, gently blow and beat, re-suspended cell, often pipe adds 5 μ l Annexin V, it is sufficiently mixed the uniform 5 μ l PI that add afterwards to mix, it is placed in the environment of room temperature lucifuge and is incubated 10min, detected with flow cytometer.

2.3.5 impact of the nitidine chloride to each phenotypic marker secretion of derived from bone marrow macrophage

2.3.5.1 the impact that ELISA method detection nitidine chloride is secreted to derived from bone marrow macrophage phenotype mark

The macrophage of bone marrow derived is collected, counted under microscope is diluted to 5 × 10 with DMEM complete mediums⁵The density of/ml, is seeded to 24 orifice plates, per 500 μ l of hole, 5%CO₂, cultivate 24h in 37 DEG C of incubators；(1) investigate optimal dosage：After cell is completely adherent, the nitidine chloride solution of variable concentrations (1 μM, 2 μM, 4 μM) is added, after 12h, add 10ng/ml LPS effect 6h.(2) investigate Best administration time：After cell is completely adherent, add the nitidine chloride solution of same concentrations, 1h, administration are simultaneously before the administration, 1h after administration, 6h after administration, 18h adds 10ng/ml LPS to act on 6h after administration, blank group (cell suspension) is set simultaneously, matched group (cell suspension+LPS), administration group (cell suspension+LPS+ variable concentrations medicines).Cell conditioned medium is collected, 1500rpm centrifugation 5min, collection supernatant are to be measured in -80 DEG C of preservations, ELISA method detection RM mark IL-10.

The macrophage of bone marrow derived is collected, as stated above inoculating cell, after cell is fully adherent, add the nitidine chloride effect 6h of variable concentrations, add 10ng/ml LPS effect 6h；Cell conditioned medium is collected, 1500rpm centrifugation 5min, collection supernatant are to be measured in -80 DEG C of preservations, the change of ELISA method detection M1 mark IL-12p70,

1.2.4 the impact that ELISA method detection nitidine chloride is secreted to RAW264.7 macrophage phenotypes mark

RAW264.7 cells are collected, counted under microscope is diluted to 5 × 10 with DMEM complete mediums⁵The density of/ml, is seeded to 24 orifice plates, per 500 μ l of hole, 5%CO₂, cultivate 4h in 37 DEG C of incubators；Dosage regimen is as follows：(1) investigate optimal dosage：After cell is completely adherent, the nitidine chloride solution of variable concentrations (1 μM, 2 μM, 4 μM) is added, after 12h, add 1 μ g/ml LPS effect 24h.(2) investigate Best administration time：After cell is completely adherent, the nitidine chloride solution of same concentrations is added, 1h, administration are simultaneously before the administration, 1h after administration, 6h after administration, 18h adds 1 μ g/ml LPS to act on 24h after administration.Blank group (cell suspension) is set simultaneously, matched group (cell suspension+LPS), administration group (cell suspension+LPS+ variable concentrations medicines) collect cell conditioned medium, 1500rpm is centrifuged 5min, collects supernatant to be measured in -80 DEG C of preservations.ELISA detects the change of rM mark IL-10.

2.3.5.2 the impact that Griess methods detection nitidine chloride is secreted to M1 marks NO

The macrophage of bone marrow derived is collected, counted under microscope is diluted to 5 × 10 with DMEM complete mediums⁵The density of/ml, is seeded to 96 orifice plates, per 120 μ l of hole, 5%CO₂, cultivate 24h in 37 DEG C of incubators；The nitidine chloride effect 6h of variable concentrations after cell is fully adherent, is added, 10ng/ml LPS effect 18h are added；Blank group (cell suspension) is set simultaneously, matched group (cell suspension+LPS), positive drug group (+LPS+50 μM of aminoguanidine of cell suspension), administration group (cell suspension+LPS+ variable concentrations medicines), takes 100 μ l of cell supernatant with isopyknic Griess reagents (A liquid：2.5% phosphoric acid solution of 1% p-aminobenzene sulfonic acid；B liquid：0.1% N-1- naphthalene ethylene diamine aqueous solutions, A:B=1:1) mix homogeneously, room temperature effect 10min, detects at microplate reader 570nm.

2.3.6 impact of the nitidine chloride to each phenotypic marker mrna expression amount of macrophage of bone marrow derived

In order to further understand the mechanism of nitidine chloride regulation and control macrophage phenotype change, using the method for Real-time PCR, impact of the nitidine chloride to each phenotypic marker mRNA level in-site of macrophage of bone marrow derived is detected from transcriptional level.

2.3.6.1 the preparation of sample

The macrophage of bone marrow derived is collected, counted under microscope is diluted to 1 × 10 with DMEM culture medium⁶The density of/ml, is seeded to 12 orifice plates, per hole 1ml, 5%CO₂, cultivate 24h in 37 DEG C of incubators；After cell is fully adherent, 4 μM of nitidine chlorides is added, after adding 10ng/ml LPS, LPS to stimulate 0h, 1h, 4h, 16h after incubation 6h, with trypsin digestion and cell, is collected in 1.5ml centrifuge tubes.

2.3.6.2 the extraction of total serum IgE

The total serum IgE in cell is extracted using the very fast extraction agent box of total serum IgE：

(1) cell for gathering each group PBS is washed once, 1500rpm centrifugation 5min, supernatant discarded.

(2) 500 μ l RA2 liquid are added in every pipe sample, gently piping and druming is until no cell mass, overturns mixing, standing 1min.

(3) sample dissociation liquid is added in sample cell (including inner sleeve and trocar sheath), in 12000rpm horizontal centrifugal 1min.

(4) inner sleeve is taken out, discards the liquid in trocar sheath, 1min is centrifuged in 12000rpm again.Repeat aforesaid operations to wash again once.

(5) inner sleeve is taken out, discards the liquid in trocar sheath, be not added with washing liquid, 12000rpm centrifugation 1min.

(6) trocar sheath is discarded, inner sleeve is put in new 1.5ml centrifuge tubes, the middle position that 25-50 μ l eluents are added to adsorbed film is drawn using pipettor, it is ensured that adsorbed film is fully infiltrated, stand 1min, 12000rpm centrifugation 1min, obtain total serum IgE.As RNA easily degrades, therefore the total serum IgE for obtaining should carry out next-step operation as early as possible, it is to avoid long-time is preserved.

2.3.6.3 RNA is quantitative and purity detecting

RNA is quantitative：Rna content is detected with Epoch ultramicron microplate spectrophotometer, 2 μ l eluents detection background is first drawn, and after background detection is qualified, 2 μ l of pipette samples RNA are added on microwell plate, two multiple holes of each sample, read OD₂₆₀/OD₂₈₀Numerical value and concentration value.

Purity detecting：

(1) OD read with Epoch ultramicron microplate spectrophotometer₂₆₀/OD₂₈₀Numerical value can embody the pollution level of RNA sample, and numerical value then illustrates that between 1.8-2.2 RNA mass is preferable.If numerical value is less than 1.8, illustrate that the pollution of the Organic substance such as albumen in RNA is more serious；If numerical value is more than 2.2, illustrate that RNA has been hydrolyzed.

(2) agarose gel electrophoresiies：1% agarose gel is prepared first, weigh 0.2g agaroses, add 20ml1 × TAE solution, mix homogeneously, heated and boiled make agarose melt completely, treat that temperature is down to 60 DEG C or so, coagulant liquid is mixed and is poured in glue groove, comb is extracted after coagulant liquid solidification by insertion comb, and addition 1 × TAE buffer did not had glass plate.After agarose gel is ready to, loading, electrophoresis is carried out, take the RNA sample of 9 μ l, add 1 μ 10 × sample-loading buffers of l, mix homogeneously loading, voltage 100V electrophoresis 20min or so.It is careful to take out gel, taken pictures with gel imaging system, observe two band of 28S and 18S, if two bands clearly become clear, and the brightness of 28S bands is almost 2 times of 18S band brightness, then prove that RNA is extracted completely and integrity is good, can be used for subsequent experimental.

2.3.6.4 reverse transcription is into cDNA

Using the PrimeScript of TaKaRa^TMRT Master Mix carry out reverse transcription, and being formulated in for reactant liquor is carried out on ice, and reaction system is as follows：

After slight mixing, in being put into PCR instrument, reverse transcription is carried out, reaction condition is as follows：

37 DEG C of 15min (reverse transcription reaction)

85 DEG C of 5sec (reverse transcription inactivation)

4℃

After reaction terminates, -20 DEG C of preservations are placed on stand-by.

2.3.6.5 Real Time PCR

Using SYBR Premix Ex Taq^TMEnter performing PCR, reactant liquor to be formulated in that carry out on ice reaction system as follows：

After slight mixing, Real Time PCR reactions in being put into PCR instrument, are carried out, reaction condition is as follows：

Solubility curve is analyzed

Used relevant primer sequence such as Table.3-1 in experiment

Table.3-1 Sequences of primers used in Real Time PCR assays

2.3.6.6 interpretation of result

In quantitative fluorescent PCR, in several circulations of the starting of amplified reaction, the variation tendency of fluorescence signal is not obvious, it is close to straight line, as baseline, a fluorescence threshold is set in the exponential phase of PCR amplifications, fluorescence signal in each sample cell is CT values from the circulation number experienced by baseline arrival threshold value, we are averagely worth to Δ CT values with the CT that the CT meansigma methodss of each sample deduct GAPDH, the sample of blank group is set to check sample, the Δ CT that check sample is deducted with the Δ CT values of other each group samples is worth to Δ Δ CT values, uses 2^{- ΔΔ CT}Represent the multiple proportion of other samples and testing gene in check sample.

2.3.7 statistical analysis

The drafting of figure is carried out using 5 softwares of Graphpad Prism.All experimental datas are represented with meansigma methodss (Mean) ± average standard error (SEM), relatively adopt Student ' s t test, P between two groups of data<0.05 is considered to have statistical significance.

2.4 experimental result

2.4.1 the macrophage purity detecting of derived from bone marrow

Experiment is marked to the macrophage of isolated derived from bone marrow with Macrophage Surface mark F4/80 and CD11b antibody, impact of the nitidine chloride to the macrophage proliferation of derived from bone marrow using FCM analysis method

This experiment have detected the impact of 1-4 μM of nitidine chloride to the macrophage proliferation of derived from bone marrow using the method for MTT.As a result show, compared with matched group, 1-4 μM of nitidine chloride does not have obvious inhibited proliferation to the macrophage of derived from bone marrow, is specifically shown in (Fig. 4).

1.4.2 impact of the nitidine chloride to the macrophage apoptosis of derived from bone marrow

Experiment have detected the impact of the nitidine chloride of variable concentrations to the macrophage apoptosis of bone marrow derived using flow cytometry (FCM).(Fig. 5) is specifically shown in, lower left region represents living cells in figure, lower right area represents viable apoptotic cell, and right regions represent late apoptic and secondary necrosis cell, and top left region represents dead cell.As a result show, compared with matched group, 1-4 μM of nitidine chloride is not all significantly affected on the macrophage ratio of the derived from bone marrow of early stage, late apoptic and death, shows that 1-4 μM of nitidine chloride can not cause the apoptosis of the macrophage of bone marrow derived.

1.4.3. impact of the nitidine chloride to each phenotypic marker secretion of macrophage of derived from bone marrow

The impact that nitidine chloride variable concentrations and different addition times are secreted to RM outstanding feature things IL-10 is studied first, as a result show, nitidine chloride (1-4 μM) can be obviously promoted the macrophages secrete IL-10 of the derived from bone marrow of LPS stimulations, the promotion rate that wherein 1,2,4 μM of nitidine chloride secrete to IL-10 respectively 21.77 ± 8.85% (p<0.05), 40.57 ± 8.44% (p<0.01), 57.80 ± 3.54% (p<0.01).Nitidine chloride stimulates 1h (1h) after (0h), LPS stimulate simultaneously to add in LPS can not all to significantly increase the secretion level of IL-10；And 1h (- 1h), front 6h (- 6h), the addition of front 18h (- 18h) nitidine chloride can significantly increase the secretion of IL-10 before LPS stimulates, its promotion rate is respectively 34.11 ± 12.61% (p<0.05), 50.20 ± 7.69% (p<0.01) He 46.45 ± 12.02% (p<0.01).Show that nitidine chloride can promote the secretion of RM mark IL-10 dose-dependant and time-dependent.

Secondly the impact that research variable concentrations nitidine chloride is secreted to M1 phenotype outstanding feature things IL-12p70 and NO, as a result show that the content of IL-12p70 and NO is significantly raised in supernatant Jing after 10ng/ml LPS stimulate 6h for the macrophage of derived from bone marrow, and 1-4 μM of nitidine chloride can substantially suppress the macrophages secrete IL-12p70 and NO of the derived from bone marrow of LPS activation, suppression ratio respectively 19.60 ± 3.08% (p that wherein 1,2,4 μM of nitidine chloride secrete to IL-12p70<0.05), 29.74 ± 2.57% (p<0.01), 40.32 ± 1.86% (p<0.01)；44.87 ± 5.22% (p are respectively to the suppression ratio of NO secretions<0.01), 51.13 ± 8.62% (p<0.01), 55.18 ± 10.61% (p<0.01), it is specifically shown in (Fig. 6)

2.4.5 impact of the nitidine chloride to each phenotypic marker mRNA expression of macrophage of derived from bone marrow

In order to confirm the impact that nitidine chloride polarizes to macrophage, further using the method for Real-time PCR studying the impact that nitidine chloride is expressed to M1 and RM phenotypic markers mRNA, as a result being displayed in LPS stimulates different time points, nitidine chloride can be different degrees of rise RM outstanding feature thing IL-10, SPHK1 and LIGHT mrna expression amount, and the mRNA expression of the proinflammatory inflammation factor IL-12p40 of M1 outstanding features and TNF-α is lowered, (Fig. 7) is specifically shown in (Fig. 8).Further demonstrate that nitidine chloride can promote conversions of the M1 to RM in vitro.

Brief summary：Nitidine chloride can promote the secretion of modulability macrophage marker IL-10, preliminary proof nitidine chloride promote the conversion of the macrophage of classical activation to modulability macrophage.Nitidine chloride can promote the expression of modulability macrophage marker IL-10, SPHK1, LIGHT in the classical activated macrophage mark IL-12p40 of the different degrees of suppression of different LPS stimulation times points, the expression of TNF-α.The above results show that nitidine chloride can promote macrophage to convert from classical activation hypotype to modulability hypotype in vitro.

The internal Pharmacodynamical mechanism research experiment of 4 nitidine chloride of embodiment

Using lumbar injection LPS inducing mouse sepsis, take Macrophage in Spleen and liver, lung tissue that sepsis mice and nitidine chloride intervene mice, the expression change of various macrophage phenotype marks is detected using the method for quantitative PCR, whether research nitidine chloride can also adjust macrophage phenotype polarization in vivo；And process the macrophage of derived from bone marrow in vitro with nitidine chloride; convert it into as modulability macrophage; using the method for cell adoptive transfer; observation gives the survival rate change of sepsis mice after drug-induced modulability macrophage; further study whether the Pharmacodynamical mechanism that nitidine chloride prevents and treats mouse sepsis, i.e. nitidine chloride play the pyemic effect of protection by regulating and controlling macrophage polarization.

Impact of the nitidine chloride to sepsis mouse spleen macrophage phenotype marker expression

1.1 experiment material

Nitidine chloride (upper Hiroad standing grain company)；

(Sigma Co., USA, from escherichia coli 055 for LPS：B5)；

0.9% normal saline (laboratory self-control)；

Biological level PBS (Hyclone companies of the U.S.)

Total serum IgE extraction agent box (Chinese Fei Jie companies)

Reverse transcription reagent box (Japanese Takara companies)

Real-time fluorescence quantitative PCR test kit (Japanese Takara companies)

1.2 laboratory animal

BALB/c mouse, male, 6-8 week old, 20-22g；There is provided by Shanghai Slac Experimental Animal Co., Ltd., raised in pharmaceutical college of The 2nd Army Medical College cleaning grade Animal House.In Animal House, keeping temperature is 21 ± 2 DEG C, 12 hours day-night cycle, and mice can ad lib drinking-water.Mice before the experiments were performed, is raised 7 days to adapt to environment.

1.3 experimental technique

1.3.1 the foundation and administration of the mouse sepsis model of LPS inductions

30 male BALB/c mouse are randomly divided into model group and administration group, and per group of 15 mices, every mouse peritoneal injection 15mg/kg LPS solution induce sepsis；Administration group 6h before lps injection gives the nitidine chloride solution of lumbar injection 10mg/kg, and model group gives the normal saline of same volume.

1.3.2 the separation and culture of the macrophage in mouse spleen source

(1) make arrangements for surgery apparatus, sterilization；

(2) mice is put to death using the method for cervical dislocation, is placed in 75% ethanol, soak 2min.Mouse peritoneal is opened with the shears for having sterilized in advance, careful separation goes out the spleen of mice, be placed in the sterilized petri dishes for filling pre-cooling PBS and wash；

(3) spleen is put on 70 μm of nylon leaching nets being placed in 1640 culture medium, spleen is rolled with the nook closing member of injector syringe, and (attention wants multiple pressure to grind less, preventing and treating damages splenocyte), nylon leaching net is rinsed with 1640 culture medium, cell is collected in 50ml centrifuge tubes, is blown and beaten repeatedly uniform；

(4) 1500rpm centrifugations 5min, careful abandoning supernatant；

(5) erythrocyte cracked liquid (compound method sees above) of 10ml is added in cell precipitation, uniform, stationary incubation 2min is blown and beaten repeatedly, the cell suspension for terminating is incubated and 5min is centrifuged in 1500rpm, careful abandoning supernatant, if now cell white then erythrocyte is abolished completely；

(6) the aseptic PBS cells of 10ml are added twice, 5min is centrifuged in 1500rpm, supernatant discarded, with 1ml RPMI-1640 culture medium re-suspended cells；

(7) cell counting, is diluted to 2 × 10 with RPMI-1640 complete mediums⁷The density of/ml, is laid in 10cm plates, each plate 10ml, 5%CO₂, cultivate in 37 DEG C of incubators.

(8) after 2 3h of cell attachment^[1], culture medium being discarded, the aseptic PBS of 5ml is added, is jiggled plate, remove not adherent cell, this step is in triplicate.

(9) aseptic PBS is added, blows and beats repeatedly plate, collect cell, as Macrophage in Spleen.

1.3.3 the impact that nitidine chloride is expressed to each phenotypic marker mRNA of macrophage that sepsis mouse spleen is originated

1.3.3.1 the preparation of sample

BALB/c mouse 1h after lps injection, lumbar injection 300mg/kg chloral hydrate anesthesias open mouse peritoneal, carefully take out spleen, as stated above isolated each group mouse spleen macrophage.

1.3.3.2 the extraction of total serum IgE

The total serum IgE in cell is extracted using the very fast extraction agent box of total serum IgE, and concrete steps refer to chapter 2 experimental section.The total serum IgE of acquisition is easily degraded, and should carry out next-step operation as early as possible, it is to avoid long-time is preserved.

1.3.3.3 RNA is quantitative and purity detecting

Specific experiment operation can be used for subsequent experimental referring to embodiment 3 if the results show RNA mass is preferable, extract completely and integrity is good.

1.3.3.4 reverse transcription is into cDNA

Using the PrimeScript of TaKaRa^TMRT Master Mix carry out reverse transcription, and specific experiment is placed on -20 DEG C of preservations stand-by referring to chapter 2 experimental section after reaction terminates.

1.3.3.5 Real Time PCR

Using SYBR Premix Ex Taq^TMEnter performing PCR, specific experiment is referring to chapter 2 experimental section.

1.3.3.6 interpretation of result

Specific experiment is operated referring to embodiment 3, uses 2^{- ΔΔ CT}Represent the multiple proportion of other samples and testing gene in check sample.

1.3.4 statistical analysis

The drafting of figure is carried out using 5 softwares of Graphpad Prism.All experimental datas are represented with meansigma methodss (Mean) ± average standard error (SEM), relatively adopt Student ' s t test, P between two groups of data<0.05 is considered to have statistical significance.

1.4 experimental result

This tests the spleen of the 1h after sepsis modeling, take model group mice and administration group mice, prepares the macrophage in spleen source, using the method detection impact of the nitidine chloride to M1 and RM mark mRNA expressions of quantitative PCR.As a result show that nitidine chloride can significantly suppress the expression (p of M1 mark IL-12p40, TNF-α, IL-6 and iNOS<0.05), and it is obviously promoted the expression (p of RM mark IL-10, SPHK1 and LIGHT<0.05).Specifically such as (Fig. 9).Brief summary：Show that nitidine chloride can promote conversion of the sepsis mouse spleen macrophage from M1 to RM in vivo.

Nitidine chloride is to sepsis Mouse Liver, the impact of macrophage in lung tissues phenotypic marker expression

2.1 experiment material

Nitidine chloride (upper Hiroad standing grain company)；

(Sigma Co., USA, from escherichia coli 055 for LPS：B5)；

0.9% normal saline (laboratory self-control)；

Biological level PBS (Hyclone companies of the U.S.)

RNAiso Plus reagents (Japanese Takara companies) reverse transcription reagent box (Japanese Takara companies)

Real-time fluorescence quantitative PCR test kit (Japanese Takara companies)

2.2 laboratory animal

BALB/c mouse, male, 6-8 week old, 20-22g；There is provided by Shanghai Slac Experimental Animal Co., Ltd., raised in pharmaceutical college of The 2nd Army Medical College cleaning grade Animal House.In Animal House, keeping temperature is 21 ± 2 DEG C, 12 hours day-night cycle, and mice can ad lib drinking-water.Mice before the experiments were performed, is raised 7 days to adapt to environment.

2.3 experimental technique

2.3.1 the foundation and administration of the mouse sepsis model of LPS inductions

10 male BALB/c mouse are randomly divided into model group and administration group, and per group of 5 mices, every mouse peritoneal injection 15mg/kg LPS solution induce sepsis；Administration group 6h before lps injection gives the nitidine chloride solution of lumbar injection 10mg/kg, and model group gives the normal saline of same volume.

2.3.2 impact of the nitidine chloride to various macrophage phenotype mark mRNA expression in sepsis Mouse Liver, lung tissue

2.3.2.1 the preparation of sample

BALB/c mouse 1h after lps injection, lumbar injection 300mg/kg chloral hydrate anesthesias, open mouse peritoneal, carefully take out its liver, lung tissue, be stored in if it can not carry out subsequent operation immediately -80 DEG C it is stand-by.2.3.2.2 the extraction of total serum IgE

The total serum IgE in tissue is extracted using the RNAiso Plus reagents of TaKaRa, and concrete operation step is as follows:

(1) liver, the lung tissue of each group mice are taken out, 30mg or so is cut with the shears for having sterilized in advance, tissue is shredded as far as possible, add 1ml RNAiso Plus reagents, it is homogenized with refiner, whole operation is carried out on ice, and homogenate is stored at room temperature 5min after terminating, in 4 DEG C of horizontal centrifugal 5min of 12000g；

(2) carefully supernatant is drawn in new EP pipes, adds the chloroform of 1/5 times of RNAiso Plus volume, firmly concussion is mixed, and is stored at room temperature 5min, in 4 DEG C of horizontal centrifugal 15min of 12000g；

(3) carefully supernatant is drawn in new EP pipes (should not carefully be drawn onto middle protein layer), adds the isopropanol of 1/2-1 times of RNAiso Plus volume, it is fully reverse to mix, 10min is stored at room temperature, in 4 DEG C of horizontal centrifugal 10min of 12000g；

(4) supernatant is carefully discarded, is precipitated with 75% ethanol purge with RNAiso Plus equivalent, in 4 DEG C of horizontal centrifugal 5min of 7500g；Net supernatant is carefully abandoned, retains precipitation；

(5) it is dried under room temperature, with appropriate DEPC water dissolving RNAs sample.

2.3.2.3 RNA is quantitative and purity detecting

RNA is quantitative：Rna content is detected with Epoch ultramicron microplate spectrophotometer, 2 μ l eluents detection background is first drawn, and after background detection is qualified, 2 μ l of pipette samples RNA are added on microwell plate, two multiple holes of each sample, read OD₂₆₀/OD₂₈₀Numerical value and concentration value.

Purity detecting：(1) OD read with Epoch ultramicron microplate spectrophotometer₂₆₀/OD₂₈₀Numerical value can embody the pollution level of RNA sample, and numerical value then illustrates that between 1.8-2.2 RNA mass is preferable.If numerical value is less than 1.8, illustrate that the pollution of the Organic substance such as albumen in RNA is more serious；If numerical value is more than 2.2, illustrate that RNA has been hydrolyzed.

(2) agarose gel electrophoresiies：1% agarose gel is prepared first, weigh 0.2g agaroses, add 20ml 1 × TAE solution, mix homogeneously, heated and boiled make agarose melt completely, treat that temperature is down to 60 DEG C or so, coagulant liquid is mixed and is poured in glue groove, comb is extracted after coagulant liquid solidification by insertion comb, and addition 1 × TAE buffer did not had glass plate.After agarose gel is ready to, loading, electrophoresis is carried out, take the RNA sample of 9 μ l, add 1 μ 10 × sample-loading buffers of l, mix homogeneously loading, voltage 100V electrophoresis 20min or so.It is careful to take out gel, taken pictures with gel imaging system, observe two band of 28S and 18S, if two bands clearly become clear, and the brightness of 28S bands is almost 2 times of 18S band brightness, then prove that RNA is extracted completely and integrity is good, can be used for subsequent experimental.

2.3.2.4 reverse transcription is into cDNA

Using the PrimeScript of TaKaRa^TMRT Master Mix carry out reverse transcription,

Being formulated in for reactant liquor is carried out on ice, and reaction system is as follows：

After slight mixing, in being put into PCR instrument, reverse transcription is carried out, reaction condition is as follows：

37 DEG C of 15min (reverse transcription reaction)

85 DEG C of 5sec (reverse transcription inactivation)

4℃

After reaction terminates, -20 DEG C of preservations are placed on stand-by.

2.3.2.5 Real Time PCR

Using SYBR Premix Ex Taq^TMEnter performing PCR

Using SYBR Premix Ex Taq^TMEnter performing PCR, reactant liquor to be formulated in that carry out on ice reaction system as follows：

After slight mixing, Real Time PCR reactions in being put into PCR instrument, are carried out, reaction condition is as follows：

Solubility curve is analyzed

Used relevant primer sequence such as Table.3-1 in experiment

Table.3-1 Sequences of primers used in Real Time PCR assays

2.3.2.6 interpretation of result

In quantitative fluorescent PCR, in several circulations of the starting of amplified reaction, the variation tendency of fluorescence signal is not obvious, it is close to straight line, as baseline, a fluorescence threshold is set in the exponential phase of PCR amplifications, fluorescence signal in each sample cell is CT values from the circulation number experienced by baseline arrival threshold value, we are averagely worth to Δ CT values with the CT that the CT meansigma methodss of each sample deduct GAPDH, the sample of blank group is set to check sample, the Δ CT that check sample is deducted with the Δ CT values of other each group samples is worth to Δ Δ CT values, uses 2^{- ΔΔ CT}Represent the multiple proportion of other samples and testing gene in check sample.

2.3.3 statistical analysis

The drafting of figure is carried out using 5 softwares of Graphpad Prism.All experimental datas are represented with meansigma methodss (Mean) ± average standard error (SEM), relatively adopt Student ' s t test, P between two groups of data<0.05 is considered to have statistical significance.

2.4 experimental result

2.4.1 impact of the nitidine chloride to various macrophage phenotype mark mRNA expression in sepsis murine liver tissue

This tests the hepatic tissue of the 1h after sepsis modeling, take model group mice and administration group mice, using impact of the method detection nitidine chloride of quantitative PCR to M1 and RM marks mRNA expression in hepatic tissue.As a result show that nitidine chloride can significantly suppress M1 characteristic indication thing IL-12p40, TNF-α, the expression (p of IL-6 and iNOS<0.05), and it is obviously promoted the expression (p of RM characteristic indication thing IL-10, SPHK1 and LIGHT<0.05).Specifically such as (Figure 10).

2.4.2 impact of the nitidine chloride to various macrophage phenotype mark mRNA expression in sepsis mouse lung tissue

This tests the lung tissue of the 1h after sepsis modeling, take model group mice and administration group mice, using impact of the method detection nitidine chloride of PCR to M1 and RM marks mRNA expression in lung tissue.As a result show that nitidine chloride can significantly suppress the expression (p of M1 mark IL-12p40, TNF-α, IL-6 and iNOS<0.05), and it is obviously promoted the expression (p of RM mark IL-10, SPHK1 and LIGHT<0.05).Specifically such as (Figure 11)

Brief summary：(1) nitidine chloride can significantly suppress the expression of M1 marks in hepatic tissue, promote the expression of RM marks.(2) nitidine chloride can significantly suppress the expression of M1 marks in lung tissue, promote the expression of RM marks.

The above results show that nitidine chloride can promote the conversion of sepsis Mouse Liver, macrophage in lung tissues from M1 to RM in vivo.Prompting nitidine chloride may be relevant with regulation and control macrophage phenotype polarization for the preventive and therapeutic effect of mouse sepsis.

Impact of the BMDMs adoptive transfers of nitidine chloride process to sepsis mouse survival rate

3.1 experiment material

Nitidine chloride (upper Hiroad standing grain company)；

(Sigma Co., USA, from escherichia coli 055 for LPS：B5)；

0.9% normal saline (laboratory self-control)；

Recombined small-mouse cytokine M-CSF (Peprotech companies of the U.S.)

Biological level PBS (Hyclone companies of the U.S.)

3.2 laboratory animal

BALB/c mouse, male, 6-8 week old, 20-22g；There is provided by Shanghai Slac Experimental Animal Co., Ltd., raised in pharmaceutical college of The 2nd Army Medical College cleaning grade Animal House.In Animal House, keeping temperature is 21 ± 2 DEG C, 12 hours day-night cycle, and mice can ad lib drinking-water.Mice before the experiments were performed, is raised 7 days to adapt to environment.

3.3 experimental technique

3.3.1 the preparation of cell sample

The macrophage with culture bone marrow derived is separated according to method previously, induction collected cell after 7 days, and counted under microscope is diluted to 1 × 10 with DMEM culture medium⁶The density of/ml, is inoculated in 6 orifice plates, per hole 2ml, 5%CO₂, cultivate 24h in 37 DEG C of incubators；After cell is fully adherent, 4 μM of nitidine chlorides are added, after adding 10ng/ml LPS, LPS to stimulate 6h after incubation 6h, with trypsin digestion and cell, PBS once, then with new PBS re-suspended cells, is collected in 15ml centrifuge tubes.

3.3.2 the foundation and administration of the mouse sepsis model of LPS inductions

30 BALB/c mouse are randomly divided into model group, nitidine chloride direct administration group and cell adoptive transfer administration group, and per group of 10 mices, every mouse peritoneal injection 15mg/kg LPS solution induce sepsis.Nitidine chloride direct administration group mice 6h before lps injection give lumbar injection 10mg/kg nitidine chloride solution；Cell adoptive transfer administration group mice 6h before lps injection is often only given tail vein injection 5 × 10⁶The macrophage of the derived from bone marrow that individual nitidine chloride is processed, model group give the normal saline of same volume.

3.3.3 the observation of mouse survival rate

After the completion of each group mice modeling, it is put back in the cage of each group, ad lib drinking-water, the survival rate observed in 84h at interval of 12h, records dead mouse number, dead mice is taken out.

3.3.4 statistical analysis

The drafting of figure and the analysis of data are carried out using 5 softwares of Graphpad Prism, the variation analyses to Kaplan-Meier survival curves are checked using log-rank, P<0.05 is considered to have statistical significance.

3.4 experimental result

In experiment, model group starts dead mouse phenomenon occur from 24h, and in dead 3 mices of 24h, now survival rate is 70%, and subsequent mice is gradually dead, and all dead to 60h, survival rate is 0；Nitidine chloride direct administration group starts 2 dead mouses occur from 48h, and now survival rate is 80%, and 48h to 84h does not have dead mouse, and survival rate is maintained at 80%；Cell adoptive transfer group starts dead mouse occur from 36h, and now survival rate is 80%, is down to 50% to 48h survival rates, and 48h to 84h does not have dead mouse, and survival rate is maintained at 50% (Figure 12).As a result show that the macrophage adoptive transfer of the derived from bone marrow that nitidine chloride is processed can significantly improve the survival rate of sepsis mice, but its protective effect is weaker than.

Brief summary：As a result show that adoptive transfer group mouse survival rate is significantly improved up to 50%, illustrate that nitidine chloride can play the pyemic effect of preventing and treating by adjusting macrophage polarization really.

Nitidine chloride impact in vivo to macrophage polarization, as a result show that nitidine chloride can significantly promote the expression of modulability macrophage outstanding feature thing IL-10, SPHK1 and LIGHT in sepsis mouse spleen macrophage and liver, lung tissue, suppress classical activated form macrophage outstanding feature thing IL-12p40, TNF-α, the expression of IL-6 and iNOS, convert to modulability macrophage so as to show that nitidine chloride promotes classical activated form macrophage in vivo.

Claims (4)

1. application of the nitidine chloride in prevention medication for treating pyemia is prepared, it is characterised in that compound of the nitidine chloride for following structural：

2. application of the nitidine chloride in treatment medication for treating pyemia is prepared, it is characterised in that compound of the nitidine chloride for following structural：

。

3. application according to claim 1 and 2, it is characterised in that the medicine is the pharmaceutical composition being made up of nitidine chloride and pharmaceutic adjuvant.

4. application according to claim 3, it is characterised in that described pharmaceutical composition is oral formulations, injection or percutaneous drug delivery preparation.