CN103705502B

CN103705502B - Application of flavonoid compound in treating inflammatory diseases

Info

Publication number: CN103705502B
Application number: CN201410009289.3A
Authority: CN
Inventors: 宋萍萍; 吴旭东
Original assignee: Institute of Botany of CAS; Nanjing University
Current assignee: Institute of Botany of CAS; Nanjing University
Priority date: 2014-01-09
Filing date: 2014-01-09
Publication date: 2017-05-24
Anticipated expiration: 2034-01-09
Also published as: CN103705502A

Abstract

The invention discloses application of flavonoid compound 5,7,3',4',5'-pentamethoxyl flavanone (PMFA) as an active ingredient for treating inflammatory diseases. The 5,7,3',4',5'-pentamethyl flavanone (PMFA) taken as an anti-inflammatory active ingredient has the characteristics of small toxicity and good treatment effect, and has an obvious inhibitory effect on inflammatory factors and especially macrophage cytokines. Thus, the 5,7,3',4',5'-pentamethoxyl flavanone (PMFA) can be taken as an anti-inflammatory preparation used for the inflammatory diseases in which other various macrophage cytokines are participated such as sepsis, arthritis, immune-type enteritis, diabetes and the like, and especially has a good treatment effect on the sepsis which is difficult to cure.

Description

A kind of application of flavone compound in diseases associated with inflammation is treated

Technical field

The invention belongs to biological pharmacy technical field, and in particular to a kind of first of flavone compound 5,7,3 ', 4 ', 5 '-five Epoxide flavanone is mainly used in the inflammation that various macrophages are participated in as the application in active treatments diseases associated with inflammation Disease property disease, such as arthritis, immunologic pattern enteritis, diabetes, especially there is good treatment to make to the pyemia for being difficult to cure With.

Background technology

Macrophage participates in the pathologic process of various solemn and just property diseases as the sincere advice constituent of natural immune system, Including pyemia, arthritis, immunologic pattern enteritis, diabetes etc..Wherein, pyemia is still a huge doctor in world wide Problem is treated, with serious systemic inflammatory syndrome, causes MOF, and cause the high death rate.In North America Country, thus it is speculated that per year over 600000, the death rate is up to 30%-50% to pathogenesis of sepsis rate.At present clinically to pyemic Treatment still lacks effective healing means.The inflammatory reaction that macrophage is participated in is considered as particularly important pathogenesis of sepsis process Factor, suppressing Macrophage Inflamatory reaction can effectively alleviate sepsis symptom, therefore target macrophage-mediated inflammation Disease can be as the screening index for the treatment of of sepsis medicine.In addition, there is macrophage at patient with rheumatoid arthritis synovial tissue Cell, secretes proinflammatory factor, growth factor, chemotactic factor (CF) etc., induces synovial membrane inflammation, causes joint injury, so regulation and control macrophage Cellular inflammation reaction can also be used as the evaluation index of anti-arthritic drugs.

Flavone compound is widely studied as the active ingredient of plurality of Chinese preparation, and its effect includes anti- Inflammation, anti-aging, anti-oxidant etc..5,7,3 ', 4 ', 5 '-pentamethoxyl flavanone, being dissolved in methyl alcohol, ethanol, acetone and chloroform etc. has Machine solvent, but on flavone compound by regulate and control macrophage function improve diseases associated with inflammation research it is also little.5,7, 3 ', 4 ', 5 '-pentamethoxyl flavanone, chemical name：5,7,3 ', 4 ', 5 '-pentamethoxyflavanone, No. CAS： 479672-30-5, molecular structure：

The content of the invention

The purpose of the present invention is exploration 5,7,3 ', 4 ', 5 '-pentamethoxyl flavanone（PMFA）As anti-inflammatory drug in purulence Application in toxication and related inflammation disease.

It is of the invention to be had shown that by research, 5,7,3 ', 4 ', 5 '-pentamethoxyl flavanone（PMFA）Mainly anti-inflammatory mechanisms are Adjustment macrophage polarization, can be used to treat the diseases associated with inflammation such as pyemia.

5,7,3 ', 4 ', 5 '-pentamethoxyl flavanone（PMFA）It is to isolate and purify that obtain or synthesize can from natural plants .PMFA does not have toxicity to normal macrophage, and this shows that PMFA may can avoid some common immunoregulation chemical combination The side effect of thing.Meanwhile, PMFA suppresses the expression of its inflammatory factor in the case where macrophage survival rate is not influenceed, and shows PMFA has preferable antiinflammatory action.Then we carry out experiment in vivo carries out detecting its anti-inflammatory efficacy.Compared with control group, PMFA can significantly improve the survival rate of mouse when the dosage of 30 mg/kg is administered, and improve lung injury.It is simultaneously scorching to serum The horizontal IL-1 β of inflammation factor, IL-6, TNF-α significantly inhibits effect.STAT1 is important transcription factor, inflammatory factor IL- 1 β, IL-6, the transcription of TNF-α are affected by it.PMFA suppresses the activation of STAT1, so as to suppress the table of downstream inflammatory factor Reach.

Beneficial effects of the present invention compared with the prior art：5,7,3 ', 4 ', 5 '-pentamethoxyl flavanone（PMFA）Come It is outstanding to inflammatory factor with small toxicity, eutherapeutic feature as anti-inflammatory active ingredient in coming from the extract of natural plants It is that macrophage cytokines significantly inhibit effect, therefore can be used for the inflammation that other various macrophages are participated in as anti-inflammatory preparation Disease property disease, such as pyemia, arthritis, immunologic pattern enteritis, diabetes, especially have good to the pyemia for being difficult to cure Therapeutic action.

Specific embodiment

Effect of the present invention is described further below by way of specific embodiment：

The PMFA toxicity detections of embodiment 1.

The cells of Raw 264.7 are with 5*10⁴Individual cells/well kind adds 100ng/ml LPS or isometric in 96 orifice plates PBS, while add PMFA37 DEG C be incubated 24 hours after, the influence that mtt assay detection compound is survived to cell line.

After the mouse macrophage strain Raw 264.7 of normal culture is incubated 24 hours jointly with the PMFA of various concentrations, Mtt assay detects its survival rate, and dosage reaches 60 μM, and cell survival rate, without substantially reduction, shows PMFA to huge compared with normal group Phagocyte strain is without toxicity.Further experiment shows that the survival rate of the macrophage that PMFA is activated to LPS does not suppress to make yet With（Figure 1B and C）.

The PMFA anti-inflammatory activities of embodiment 2. are detected

The cells of Raw 264.7 are with 1*10⁶Individual cells/well kind is in 6 orifice plates, while adding PMFA and 100 ng/ml fat Polysaccharide（LPS）After 37 DEG C are incubated 6 hours, QPCR methods detect the change of IL-1 β, IL-6 mRNA levels.

Experimental result shows：After LPS stimulates, macrophage is activated, cell factor IL-1 β, IL-6 mRNA level liters It is high（Fig. 1 D and E）.And under PMFA effects, IL-1 β, IL-6 mRNA level in-site are significantly reduced（Fig. 1 D and E））.Complex chart 1B's and C As a result, show, PMFA suppresses macrophage activation on the premise of not influenceing macrophage to survive, disclose PMFA to inflammation because Especially macrophage cytokines significantly inhibit effect to son, therefore can be used for other various macrophages participations as anti-inflammatory preparation Diseases associated with inflammation, such as pyemia, arthritis, immunologic pattern enteritis, diabetes.

The PMFA of embodiment 3. is to pyemic effect

1）The induction of mouse sepsis

Take 6-8 week old C57/BL6 mouse, 100 microlitres of 10mg/kg lipopolysaccharides of intraperitoneal injection（LPS）, normal group mouse note 100 microlitres of PBS are penetrated as control.

2）The observation of mouse survival rate

2 hours before injection LPS, to 100 microlitres of PMFA of mouse peritoneal injection olive oil dissolving, normal group and model group abdomen 100 microlitres of olive oil are injected as control in chamber.Observed every 4 hours and record mouse survival rate, until mouse survival rate is steady It is fixed no longer to change.

3）Serum levels of inflammatory cytokines IL-1 β, IL-6, the detection of TNF-α

Separately take 2 hours before a collection of mouse injection LPS, the 100 microlitres of PMFA dissolved to mouse peritoneal injection olive oil, normally Group and 100 microlitres of olive oil of model group intraperitoneal injection are as control.By sacrifice after 4 hours, pluck eyeball and take blood, 37 °C of water 3500 rpm are centrifuged 15 minutes after bath 30min, draw serum ELISA methods detection inflammatory factor IL-1 β, IL-6, TNF-α water Flat

4）The observation of lung tissue pathological change

The mouse of execution takes lung tissue simultaneously.A part of 10% formalin solution is fixed, and FFPE makees 4-5 micro- The thick coronal section of rice, and use stained with Hematoxylin-Eosin.Tissues observed lesion and inflammatory cell infiltration.

5）The detection of lung tissue cytokine mRNA levels

The mouse of execution, total serum IgE is extracted after taking a part of lung tissue plus Trizol homogenate, and 1g RNA reverses are taken after quantifying CDNA is recorded into, reaction system is as follows：1 μ g total serum IgEs, 2000 pM Oligo dT, 1.0 mM dNTP, 200 U reverses Record enzyme, 5 × transcription buffer.Full-length genome cDNA is expanded using specific primer, and reaction system is as follows：1 μl CDNA, 0.5 μM of upstream and downstream primer, 5 μ l iQ SYBR Green permix.PCR primer and Amplification are shown in Table 1.Expand Increase result to be analyzed using BioRad CFX manager software.

The parameter of table 1 each cell factor

6）The detection of lung tissue p-STAT1 levels

The mouse of execution, total protein is extracted after taking a part of lung tissue plus cell pyrolysis liquid homogenate, and 25g is taken after quantifying Albumen carries out SDS-PAGE electrophoretic separation, and trace is transferred to pvdf membrane, and 5% skim milk room temperature is closed 1 hour, with p-STAT1 Or 4 °C of overnight incubations of STAT1 antibody, it is the antibody for combining to wash film and remove, and is incubated 2 hours with corresponding secondary antibody, washes film, is developed.

Result shows：

The PMFA of mouse peritoneal injection various dose（15mg/kg, 30mg/kg）Pneumoretroperitoneum injects LPS within 2 hours.LPS is lured Hair mouse produces serious acute inflammatory reaction, serum levels of inflammatory cytokines to raise, and multiple organs occur lesion, with death higher Rate.Compared with model group, PMFA high dose groups（30 mg/kg）Mouse survival rate is significantly raised.Meanwhile, short-term experiment result table Bright, PMFA treatment groups mice serum inflammatory factor IL-1 β, IL-6, TNF-α level is suppressed significantly（Fig. 2）.Illustrate that PMFA can Significantly improve the pyemia mouse state of an illness.

Under LPS effects, mouse multiple organ is damaged, and wherein lungs sustain damage at first.After LPS is acted on 4 hours, mouse lung Dirty tissue morphology is destroyed, and non-viable non-apoptotic cell increases, and with obvious inflammatory cell infiltration.And the therapeutically effective guarantors of PMFA Mouse lung is protected.Wherein, lesion tissue is eased, and non-viable non-apoptotic cell, the inflammatory cell of infiltration are reduced.Meanwhile, lung tissue The cellular inflammation factor IL-1 β, IL-6, TNF-α mRNA level are suppressed, and have organized destruction of the inflammatory factor to internal organs.Say Bright PMFA can significantly improve pyemia mouse lung lesion（Fig. 3）.

STAT1 is important transcription factor, and inflammatory factor IL-1 β, IL-6, the transcription of TNF-α is affected by it.LPS Under stimulation, STAT1 is phosphorylated（Its activated form）And nucleus is indexed into from endochylema, and combined with specific DNA fragmentation, open The transcription of dynamic corresponding gene.The STAT1 of the mouse lung activated form that LPS stimulates increases.Immunohistochemical assay result table Bright, after LPS effects, STAT1 more appears in nucleus, exercises its functional transcription.In PMFA treatment groups mouse lung core STAT1 is significantly reduced（Fig. 4 A）.Meanwhile, western blot test result indicate that, PMFA treatment groups mouse lungs homogenate egg White STAT1 phosphorylation levels reduction（Fig. 4 B）.These results indicate that PMFA suppresses the activation of STAT1, so that it is scorching to suppress downstream The expression of inflammation factor.

Brief description of the drawings

Fig. 1：PMFA does not influence the survival rate of macrophage, while suppressing the activation of macrophage.

（A）HPLC methods detect PMFA purity.

（B）The cells of Raw 264.7 are with 5*10⁴Individual cells/well kind adds PBS to add PMFA37 DEG C simultaneously in 96 orifice plates After being incubated 24 hours, the influence that mtt assay detection compound is survived to cell line.

（C）The cells of Raw 264.7 are with 5*10⁴Individual cells/well kind adds 100 ng/ml lipopolysaccharides in 96 orifice plates （LPS）After adding PMFA37 DEG C to be incubated simultaneously 24 hours, the influence that mtt assay detection compound is survived to cell line.

（D, E）The cells of Raw 264.7 are with 1*10⁶Individual cells/well kind is in 6 orifice plates, while adding PMFA and 100 ng/ After 37 DEG C of ml LPS are incubated 6 hours, QPCR methods detect the change of IL-1 β, IL-6 mRNA levels.Result with mean ± S.e.m is represented.^*P<0.05, ^**P<0.01, vs LPS groups.

Fig. 2：PMFA improves mouse sepsis.

（A）The PMFA of mouse peritoneal injection various dose（15mg/kg, 30mg/kg）Pneumoretroperitoneum injects LPS within 2 hours.See Examine and record mouse survival rate.N=12,^**P<0.01, vs LPS groups.

（B）The PMFA of mouse peritoneal injection various dose（15mg/kg, 30mg/kg）Pneumoretroperitoneum injects LPS within 2 hours.Four By sacrifice after hour, pluck eyeball and take blood, ELISA method detection serum levels of inflammatory cytokines IL-1 β, IL-6, TNF-α level.n= 6, as a result represented with mean ± s.e.m.^*P<0.05, ^**P<0.01, vs LPS groups.

Fig. 3：PMFA improves pyemia mouse lung lesion.

（A）The PMFA of mouse peritoneal injection various dose（15mg/kg, 30mg/kg）Pneumoretroperitoneum injects LPS within 2 hours.Four By sacrifice after hour, the formalin solution of a part of lung tissue 10% is fixed, FFPE, makees 4-5 microns of thick hat Shape is cut into slices, and uses stained with Hematoxylin-Eosin.Tissues observed lesion and inflammatory cell infiltration.

（B）A part of lung tissue adds Trizol that total serum IgE, QPCR methods detection IL-1 β, IL-6 TNF-αs are extracted after being homogenized The change of mRNA level in-site.N=6, is as a result represented with mean ± s.e.m.^*P<0.05, ^**P<0.01, vs LPS groups.

Fig. 4：PMFA suppresses lung tissue STAT1 activation.

（A）The PMFA of mouse peritoneal injection various dose（15mg/kg, 30mg/kg）Pneumoretroperitoneum injects LPS within 2 hours.Fig. 3 （A）Through dewaxing, antigen retrieval with p-STAT1 antibody hybridizations after the treatment such as closing, and uses Real for the section of being unstained for making Envision Detection kit detect that it is expressed.

（B）After separately taking a part of lung tissue addition cell pyrolysis liquid homogenate, western blot methods detection p-STAT1 Expression.

Claims

1.5,7,3 ', 4 ', 5 '-pentamethoxyl flavanone is used to prepare treatment diseases associated with inflammation septicopyemia as sole active agent Application in disease drug.