CN112121042B

CN112121042B - Application of PSO in preparation of anti-septicemia and myocardial damage drug induced by anti-septicemia

Info

Publication number: CN112121042B
Application number: CN202011002459.7A
Authority: CN
Inventors: 杨阳; 邓超; 赵琳; 齐亚婷; 杨雯雯; 姬婷; 尚佳佳; 吴振; 丁宝平
Original assignee: Northwest University
Current assignee: Northwest University
Priority date: 2020-09-22
Filing date: 2020-09-22
Publication date: 2021-11-16
Anticipated expiration: 2040-09-22
Also published as: CN112121042A

Abstract

The invention discloses application of psoralen in preparing a medicament for treating and/or preventing septicemia and myocardial damage induced by the septicemia. The research of the invention finds that: PSO increases sepsis survival, improves the sepsis score in mice, decreases the number of leukocytes, lymphocytes, monocytes, granulocytes, and decreases the levels of lactate dehydrogenase, urea nitrogen, creatine kinase, aspartate aminotransferase; PSO has functions of improving cardiac contraction function and maintaining normal shape of myocardial tissue; in addition, PSO can inhibit myeloperoxidase and interleukin-1 beta high expression after myocardial damage induced by septicemia, reduce mRNA level of inflammation related molecules IL-1 beta, IL-6 and NOD-like receptor family 3, and reduce superoxide anion accumulation caused by septicemia, thereby playing anti-inflammatory and anti-oxidative stress effects.

Description

Application of PSO in preparation of anti-septicemia and myocardial damage drug induced by anti-septicemia

Technical Field

The invention relates to a new indication of Psoralen (PSO), in particular to application of PSO in preparing a medicament for resisting septicemia and myocardial damage induced by the septicemia.

Background

Psoraleadin PSO has a structural formula shown in formula I, and has various pharmacological activities including antioxidant, antibacterial, antiinflammatory, antidepressant and estrogen-like effects.

Disclosure of Invention

The inventor discovers the following indexes of survival rate, blood routine, blood biochemistry, inflammation, oxidative stress and myocardial damage by constructing a septicemia and myocardial damage animal model induced by the septicemia:

PSO can increase sepsis survival; improving a sepsis score; reducing the number of White Blood Cells (WBC), Lymphocytes (LYM), Monocytes (moncytes, MON), granulocytes (granulate, GRA) and reducing the levels of Lactate Dehydrogenase (LDH) Urea Nitrogen (BUN), Creatine Kinase (CK), Aspartate Aminotransferase (AST);

at the same time, PSO improves myocardial tissue damage and cardiac contractile function due to sepsis, at least by increasing the left ventricular end systolic Volume (Volume; s) and the reduction of the left ventricular end diastolic Volume (Volume; d); and decrease the increase of the left ventricular post-systolic wall thickness (LVPW; s), the left ventricular post-diastolic wall thickness (LVPW; d) and the Heart Rate (HR), thereby achieving the effect of improving the systolic function of the Heart;

PSO can also inhibit the high expression of Myeloperoxidase (MPO), Interleukin-1 beta (Interleukin-1 beta, IL-1 beta), Ly6c and F4/80, reduce the mRNA level of IL-1 beta, IL-6 and NOD-like receptor family 3(NOD-like receptor hormone-binding protein 3, NLRP3) after heart muscle tissue injury induced by septicemia, and reduce the accumulation of superoxide anions caused by septicemia, thereby playing the roles of anti-inflammation and anti-oxidative stress;

based on the discovery, the invention provides application of PSO in preparing a medicine for treating and/or preventing septicemia and myocardial damage induced by the septicemia.

Meanwhile, the medicine for treating and/or preventing septicemia is prepared from PSO and pharmaceutical excipients.

Further, the medicine is an intravenous injection preparation.

Further, the drug is administered in a dose of 12.5mg to 50mg PSO per kg body weight.

Description of the drawings:

fig. 1 is the influence of PSO on the survival rate of mice after CLP injury, and the survival conditions of each group of mice within 72h after CLP operation are observed, wherein a graph is a mouse survival rate modeling picture, a graph B is a mouse survival rate curve, the result is represented by mean ± standard deviation, and n is 12;^*sham group, P<0.05；^#CLP group, P<0.05；

Fig. 2 shows the influence of PSO on the score of the mouse sepsis after CLP injury, and the mouse post-injury state is scored according to sepsis score related indexes at 8h after the mouse surgery, wherein a graph is a mouse functional test modeling picture, a graph B is a sepsis score result, the result is represented by mean ± standard deviation, and n is 8;^*sham group, P<0.05；^#Clp group;

fig. 3 shows the effect of PSO on various indices of mouse blood routine after CLP injury, blood routine was measured 8h after mouse surgery, the results are expressed as mean ± standard deviation, and n is 6;^*sham group, P<0.05；^#Clp group;

fig. 4 shows the effect of PSO on various indicators of blood biochemistry of mice 8h after CLP injury, and the blood routine was measured 8h after mice surgery, and the results are expressed as mean ± standard deviation, where n is 6;^*sham group, P<0.05；^#Clp group;

FIG. 5 shows the effect of PSO on the morphology of myocardial tissue 8h after CLP injury, and the results of HE staining of myocardial tissue sections; HE, hematoxylin-eosin;

FIG. 6 shows the effect of PSO on cardiac function indexes after CLP injury for 8h, and echocardiography detection is performed for 8h after mouse operation, wherein A is a statistical analysis chart of a left ventricle long axis section, an M-mode typical picture and various cardiac function indexes beside a sternum of an echocardiography, B is a statistical analysis chart of an echocardiography short axis section, an M-mode typical picture and various cardiac function indexes, and the resultExpressed as mean ± standard deviation, n is 6,^*sham group, P<0.05；^#CLP group, P<0.05; left ventricular end-systolic volume; left ventricular end diastolic volume; left ventricular end-systolic wall thickness; left ventricular end-diastolic wall thickness; heart rate;

FIG. 7 is a graph showing the effect of PSO on myocardial inflammation-related markers 8h after CLP injury, immunohistochemical staining results of typical inflammation-related markers MPO and IL-1 β, and statistical analysis; inflammation-related molecules IL-1 beta, IL-6, NLRP3 mRNA expression level; wherein, the graph A is the result of immunohistochemical staining, the graph B is the result of mRNA expression level, the result is expressed by mean + -standard deviation, n is 6,^*sham group, P<0.05；^#CLP group, P<0.05；

Fig. 8 is a graph showing the effect of PSO on the level of superoxide anion in myocardial tissue 8h after CLP injury, myocardial tissue DHE staining results and statistical analysis, with mean ± sd, and n-6.^*Sham group, P<0.05；^#CLP group, P<0.05, DHE, superoxide anion fluorescent probe.

Detailed Description

Sepsis refers to a systemic infectious syndrome in which various pathogenic bacteria invade the blood circulation and rapidly grow and multiply in the blood to produce a large amount of toxins and metabolites, resulting in severe toxemia. Exacerbation of sepsis can cause septic shock, disseminated intravascular coagulation and impaired organ function, severely threatening the life of the patient. Heart is one of the target organs vulnerable to sepsis, and there is ample evidence that sepsis can lead to cardiac dysfunction. Clinical studies show that mortality of patients with septicemia complicated with cardiac dysfunction is as high as 70% -90%. While mortality in septic patients without cardiac dysfunction is about 20%.

The invention discloses a CLP (common Ligation and perforation) animal model, which is a classic septicemia animal model.

The heart muscle damage induced by septicemia belongs to one of infectious heart muscle damage, which refers to reactions such as cardiac enlargement, heart failure, cardiogenic shock or abnormal heart rhythm in the virus infection process or recovery period, and typical symptoms are fatigue, weakness, inappetence, nausea, vomiting, dyspnea, pale complexion, fever and the like.

The infectious myocardial injury of the invention particularly refers to myocardial inflammatory lesions caused by bacteremia caused by bacterial endocarditis, systemic or other organ infection and myocardial inflammatory lesions caused by sepsis caused by systemic or other organ infection, and adrenal cortical hormone and broad-spectrum antibiotics are used clinically to prevent dysbacteriosis. During the application, the presence or absence of fungal infections of the digestive, urinary and respiratory tracts should be observed with great care.

The invention is further illustrated by the following examples. The present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

The PSO purity used in the following examples was 98% or more by HPLC. The animals are purchased from the fourth university of military medical science laboratory animal center, and the reagents are purchased in the market. Unless otherwise specified, the experimental methods or related detection methods used in the following examples employ methods known in the art.

Example 1 study by the inventors found that PSO can ameliorate mouse death caused by sepsis

The scheme is as follows:

constructing a sepsis model at the in vivo level by using CLP operation, and giving PSO pre-protection.

The method comprises the following steps:

wild-type BALB/c mice were used as study subjects, and were grouped by a random number table method according to study design, and mouse infectious myocardial injury models were replicated from severe infectious myocardial injury models according to CLP experimental methods published by Rittirsch D et al. The specific experimental steps are as follows:

(1) grouping: survival rate experiments: BALB/c mice were divided into sham, CLP, PSO groups (12.5, 25, 50mg/kg dose), 12 per group;

(2) administration: performing pre-protection treatment (intraperitoneal injection) 6 days before operation, and feeding DMSO (dimethyl sulfoxide) in a sham operation group and a CLP group according to the concentration of 1ml/kg (body weight), and PSO (dissolved in DMSO to prepare PSO solutions with the dosages of 12.5mg/kg, 25mg/kg and 50mg/kg respectively) in a protection group for 1 time every 2 days and 3 times in total, so as to ensure that the time period of each administration is the same as the operation time;

(3) molding: model making on day 7, and anaesthetizing the mice by adopting a small animal inhalation anesthesia system: the mouse inhales oxygen containing isoflurane 2% (volume fraction vol/vol), the anesthesia flow is 3L/min, the flow in the molding process is 1.5L/min, the anesthesia degree monitoring standard is that withdrawal reflex of limbs disappears, the mouse is fixed and continuously inhales oxygen containing isoflurane 2% to maintain anesthesia; secondly, preparing skin in the middle area of the abdomen of the mouse, disinfecting the skin twice by 75% ethanol, extending the middle of the lower abdomen and making a longitudinal incision for 1cm, incising and separating the skin and subcutaneous tissues layer by layer to see a leucorrhea line, incising the rectus abdominis and peritoneum by extending the leucorrhea line, wetting the two sides of the incision by 0.9% normal saline, entering the abdomen by using a bending forceps, finding the cecum and pulling out gently, gently squeezing the excrement close to the ileocecal valve to the tail end of the cecum (avoiding air residue), ligating the cecum from the tail end of the cecum to the 2/3 position on the connecting line of the ileocecum (survival rate test is an aggravated model, performing functional test by adopting 1/3 parts of ligation models from the tail end of the cecum to the connecting line of the ileocecum, and the rest steps are the same as the aggravated model) and ligating the cecum by using a No. 4 sterile surgical suture, penetrating the ligated intestines (blood vessel) by using a 2.5ml syringe needle at the middle point of the ligating line and the tail end of the cecum, gently squeezing the cecum after penetrating, and allowing the cecum contents of the cecum to flow out along the puncture hole, bringing the cecum and all the surrounding intestinal canals into the abdominal cavity, and suturing the peritoneum and the skin layer by using a No. 3 sterile operation suture; ③ after the operation, all the experimental mice are injected with physiological saline (10ml/kg body weight) with 37 ℃ on the back for liquid recovery immediately after the operation, and then the mice are returned to the mouse cage after being marked to wait for revival. The sham group was identical to the experimental group except that cecal ligation and perforation were not performed.

(4) Timing is started after CLP operation, observation is carried out once every 1h, the death number of each group of mice within 72h is recorded, and the survival rate is counted and analyzed;

(5) according to the survival rate results of each group, after the appropriate concentration of PSO under the scheme of the embodiment is selected, the animal experiment is carried out in the same way, and the obtained specimen is subjected to subsequent detection.

As a result:

the mouse survival rate curve is shown in fig. 1B, compared with the control group, the survival rate of the mouse after CLP treatment is 40% (P <0.05) for 72h, the survival rate of the mouse after PSO (12.5mg/kg) treatment is about 70% (P <0.05), the survival rate of the mouse after PSO (25mg/kg) treatment is about 70% (P <0.05), and the survival rate of the mouse after PSO (50mg/kg) treatment is about 80% (P <0.05), which indicates that PSO can increase the survival rate of the mouse after CLP and the optimal PSO protective concentration is 50mg/kg (the optimal protective concentration is adopted in the subsequent functional tests).

Example 2 research by the inventors found that PSO can improve sepsis score and blood routine changes caused by sepsis.

The scheme is as follows:

a CLP operation is used to construct a model of sepsis and its induced myocardial damage at the in vivo level (all subsequent functional tests are performed by ligating the cecum end to 1/3 on the ileocecal valve line, and the rest steps are the same as the aggravation model, see fig. 2A), and PSO treatment is given.

The method comprises the following steps:

(1) mice 8h after CLP were scored according to a sepsis scoring table, using the following literature disclosures:

[1]Nemzek JA,Hugunin KM,Opp MR:Modeling sepsis in the laboratory:merging sound science with animal well-being.Comp Med 2008,58:120–128.

[2]Huet O,Ramsey D,Miljavec S,Jenney A,Aubron C,Aprico A,Stefanovic N,Balkau B,Head GA,de Haan JB,Chin-Dusting JP:Ensuring animal welfare while meeting scientific aims using a murine pneumonia model of septic shock.Shock(Augusta,Ga)2013,39:488–494.

[3]Langford DJ,Bailey AL,Chanda ML,Clarke SE,Drummond TE,Echols S,Glick S,Ingrao J,Klassen-Ross T,Lacroix-Fralish ML,Matsumiya L,Sorge RE,Sotocinal SG,Tabaka JM,Wong D,van den Maagdenberg AM,Ferrari MD,Craig KD,Mogil JS:Coding of facial expressions of pain in the laboratory mouse.Nature methods 2010,7:447–449.

[4]Bradly Shrum,Ram V Anantha,Stacey X Xu,Marisa Donnelly,SM Mansour Haeryfar,John K McCormick:A robust scoring system to evaluate sepsis severity in an animal model.BMC Research Notes 2014,7:233。

(2) detecting the change of the conventional indexes of the blood of the mice 8h after CLP operation: after 8h of injury, blood is taken by an eyeball-picking blood-taking method, and blood routine detection is carried out by a full-automatic blood routine instrument.

As a result:

the mice are scored for septicemia after being treated for 8 hours by CLP, and the result is shown in figure 2, compared with the control group, the score of the septicemia of the CLP group is obviously increased, and the score of the septicemia is obviously reduced after being protected by PSO;

changes in blood routine-related indices were measured 8h after CLP treatment in mice, as shown in fig. 3, with a significant increase in WBC, LYM (P <0.05), and a significant decrease in both WBC, LYM (P <0.05) but no significant effect on RBC and PLT (P >0.05) compared to control.

Example 3 research by the inventors shows that PSO can improve biochemical changes in blood caused by septicemia

The scheme is as follows:

a CLP operation is adopted to construct a model of septicemia and myocardial damage induced by the septicemia at the in vivo level, and PSO treatment is given.

The method comprises the following steps:

detecting the change of various indexes of blood biochemistry of the mouse 8h after CLP operation: after 8h of injury, blood is taken by an eyeball-picking blood-taking method, whole blood of each group is collected, 3000r/min is carried out, centrifugation is carried out for 10min, serum is sucked, and then detection is carried out by using a full-automatic blood biochemical analyzer.

As a result:

changes in blood biochemical-related indices were measured 8h after CLP treatment in mice, and as shown in fig. 4, levels of LDH, BUN, CK, and AST were all significantly increased (P <0.05) in serum 8h after CLP injury and significantly decreased (P <0.05) after PSO treatment, compared to control groups.

Example 4 research by the inventors found that PSO ameliorates myocardial tissue damage caused by sepsis.

The scheme is as follows:

The method comprises the following steps:

(1) and (3) carrying out HE staining on myocardial tissue:

embedding paraffin: slowly injecting normal saline containing heparin into the apex of the heart, and replacing the perfusion liquid with 4% paraformaldehyde stationary liquid when the liquid flowing out from the right auricle becomes transparent; after the paraformaldehyde tissue is successfully fixed, cutting off each blood vessel along the root of the heart, and completely taking down the heart; after the heart is taken out, cutting off the left half (left atrium and left ventricle) of the heart, putting the cut left half into 4% paraformaldehyde, and performing after-fixation for at least 24 h; sequentially soaking in 80%, 95% and 100% ethanol for 40min, and soaking in mixed solution of 100% ethanol, 100% ethanol and xylene at a ratio of 1: 1 for 30min to dehydrate and remove water; then dipping wax for 3h in an embedding machine, and finally dropping wax for embedding.

Cutting into slices: the thickness of the slice is set to be 5 mu m, the slice is pasted on a polylysine film-coated glass slide by using a slice dragging method, and after the slice is baked for 1h at 70 ℃, the slice is baked for 5h at 60 ℃.

③ dyeing: soaking the slices in xylene for 10min, replacing xylene, soaking for 10min again, sequentially soaking for 2min to remove wax to water according to the sequence of 100%, 95%, 80% ethanol and deionized water for dyeing; immersing the slices in hematoxylin dye solution for dyeing for 3min, and washing with tap water for 5 min; soaking in 1% hydrochloric acid ethanol for 30s, decolorizing with 1% ammonia water for 3min, and washing with tap water for 3 min; immersing in eosin dye solution for dyeing for 3min, and washing with tap water for 3 min; soaking for 2min according to 70% ethanol, 80% ethanol 30s, 95%, 100% gradient ethanol, xylene, and xylene respectively, and dehydrating for transparency; and (5) sealing the neutral gum.

As a result:

the HE staining result of mouse myocardial tissue shows that, as shown in fig. 5, compared with the control group, the CLP damaged myocardial tissue has disordered structure, the number of nuclei in the visual field is significantly increased, and the infiltration of microvascular mononuclear cells among the myocytes is increased; compared with CLP group, HE staining after PSO shows that the myocardial tissue structure is relatively clear, the number of cell nucleuses in visual field is reduced, and the infiltration of microvascular mononuclear cells among myocytes is reduced.

Example 5: the inventor researches and discovers that PSO can improve myocardial function damage caused by septicemia

The scheme is as follows:

The method comprises the following steps:

ultrasonic detection of mouse heart function 8h after CLP operation by using animal: the method comprises the following steps that the fur of the left chest area of a mouse is removed one day before ultrasonic detection of each group of animals, the mouse is anesthetized by 2% isoflurane, the flow of anesthetic gas is 1L/min, the isoflurane is fixed on a thermostatic plate at 37 ℃ after being anesthetized by inhalation, the left thorax is fully exposed, a 30MHz probe is adopted, a standard long-axis section of the left ventricle beside the sternum and a standard short-axis section of the papillary muscle of the left ventricle are selected, M-mode cardiac ultrasonic section images are recorded, and measurement indexes comprise: left ventricular end systolic volume, left ventricular end diastolic volume, left ventricular end systolic back wall thickness, left ventricular end diastolic back wall thickness, heart rate, and the like.

As a result:

the results of ultrasonic detection of the myocardial contractility of the mice 8h after the CLP operation by the animals are shown in fig. 6A (the ultrasonic result of the long axis of the left ventricle beside the sternum) and 6B (the ultrasonic result of the short axis of the left ventricle), compared with the control group, the left ventricular end systolic volume and the left ventricular end diastolic volume of the hearts of the mice are significantly reduced (P <0.05), and the cardiac function is significantly improved (P <0.05) after PSO protection; compared with the control group, the wall thickness of the mouse after the left ventricular systole end and the wall thickness of the mouse after the left ventricular diastole end are obviously thickened, the heart rate is obviously accelerated (P is less than 0.05), and the heart function is obviously improved (P is less than 0.05) after the PSO protection is given.

Example 6: the inventor researches and discovers that PSO improves myocardial damage caused by infectious CLP by reducing inflammatory reaction.

The scheme is as follows:

The method comprises the following steps:

(1) immunohistochemical detection:

the steps of paraffin embedding and slicing are the same as above;

dyeing: slices were dewaxed conventionally to water: taking paraffin sections of mouse heart tissue of each group, sequentially passing through xylene for 2 times, 10min each time, 100% ethanol for 2 times, 10min each time; respectively soaking 95%, 90%, 80%, and 70% ethanol for 5min 1 time, and soaking in distilled water for 5 min; antigen retrieval: performing microwave antigen retrieval for 20min by using a sodium citrate buffer solution, and washing for 10min by using running water; blocking endogenous peroxidase: 3% hydrogen peroxide, room temperature 15 min. Washing with PBS for 5min for 3 times; and (3) sealing: dropwise adding 5% normal goat serum confining liquid, and incubating at room temperature for 60 min; dropping primary antibody: excess serum was wiped off, primary antibody was added and incubated overnight at 4 ℃. Washing with PBS for 5min for 3 times; and (4) dropwise adding a secondary antibody: dripping horseradish peroxidase HRP-labeled secondary antibody (1: 5000, prepared by PBS), incubating for 1h in an incubator at 37 ℃, washing for 3 times by PBS, and washing for 5min each time; DAB color development: dripping DAB for 0.5-3min, controlling color development degree under microscope, washing with flowing water for 10min, counterstaining with hematoxylin, differentiating with 1% hydrochloric acid alcohol, decolorizing with 1% ammonia water, dehydrating, clearing with xylene, and sealing with neutral gum.

Observation and photographing under a microscope: observing and taking a picture under a microscope, taking a positive staining part in a brown yellow granular deposition area of the tissue section under a light microscope, randomly finding 20-30 non-overlapping visual fields for each section, and performing semi-quantitative calculation by adopting medical Image analysis software Image J5.0 software.

(2) Real-time quantitative fluorescent PCR

Extracting total RNA of a sample: taking the centrifuge tube filled with the mouse heart sample out of liquid nitrogen, adding 1mL of Trizol and 2 grinding beads, placing the centrifuge tube on a tissue disruptor to disrupt at 60Hz for 1min, slightly shaking the centrifuge tube to uniformly mix Trizol and the sample, and placing the mixture on ice to disrupt for 5 min; adding 200 μ L chloroform, shaking the tube body manually and vigorously for 15s, incubating at room temperature for 15min, and centrifuging at 4 deg.C for 15min in a high-speed centrifuge at 12000 rmp/min; at the moment, the RNA is completely positioned in the upper aqueous phase, a pipettor is used for sucking 400 mu L of aqueous phase, transferring the aqueous phase into a clean centrifugal tube without RNase, adding isopropanol with the same volume for mixing, manually shaking for 15s, incubating at room temperature for 10min, and then placing in a high-speed centrifuge at 4 ℃ for centrifugation for 15min, wherein the rotating speed is 12000 rmp/min; discarding the supernatant, adding 75% ethanol (prepared with DEPC water), manually shaking to suspend the RNA precipitate, incubating at room temperature for 5min, centrifuging at 4 deg.C and 8000rmp/min for 5min, and repeating the operation once; discarding the ethanol solution, leaving RNA precipitate, and drying at room temperature for 5-10 min; dissolving the RNA precipitate with 20 μ L DEPC water, opening the DNA/RNA concentration meter, measuring the concentration of the sample, and storing in a refrigerator at-80 deg.C.

Reverse transcription: the extracted total RNA was taken out from a refrigerator at-80 ℃, added with a reverse transcription reagent (purchased from Aikery bioengineering Co., Ltd., Hunan, Cat. No.: AG11706) and placed in a PCR instrument for reverse transcription, and the reverse transcription procedure was as follows: 15min at 37 ℃; 5s at 85 ℃; the inverted cDNA was stored at-20 ℃ and the reverse transcription system is shown in Table 1:

TABLE 1 reverse transcription System

③ qRT-PCR: taking the cDNA, the kit (purchased from Hunan Aikorui bioengineering Co., Ltd.; product number: AG11409-S) and the required primers (purchased from Shanghai Bioengineering bioengineering Co., Ltd.; NLRP 3: forward primer 5'-GGAGTTCTTCGCTGCTATGTA-3'; reverse primer 5'-GGACCTTCACGTCTCGGTTC-3'; IL-1. beta. forward primer 5'-GTGTCTTTCCCGTGGACCTT-3'; reverse primer 5'-CATCTCGGAGCCTGTAGTGC-3'; IL-6: forward primer 5'-TTGGGACTGATGCTGGTGAC-3'; reverse primer 5'-GGTATAGACAGGTCTGTTGGGAGT-3') out of a refrigerator at-20 ℃ to prepare the equipment required for the experiment; after the sample and the kit are melted, preparing a solution according to the following proportion, and performing an experiment by using a program set by an RT-qPCR instrument; the reaction conditions are as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 15 s; annealing and extending for 20s at 58 ℃; final extension at 72 ℃ for 30s, and the reaction systems are shown in tables 2-1, 2-2 and 2-3:

TABLE 2-1 qRT-PCR reaction System

TABLE 2-2 qRT-PCR reaction System

TABLE 2-3 qRT-PCR reaction System

As a result:

the IHC staining result of mouse myocardial tissues shows that as shown in FIG. 7A, compared with a control group, the expressions of MPO and IL-1 beta are obviously increased after CLP injury, and the expression quantity of the CLP treated with PSO is obviously reduced;

the qRT-PCR results of mouse myocardial tissues show that, as shown in FIG. 7B, the mRNA levels of inflammation-related molecules IL-1 beta, IL-6 and NLRP3 are remarkably increased (P <0.05) after CLP injury and the expression levels are reduced (P <0.05) after PSO treatment compared with the control group.

Example 7: the inventors have found that PSO ameliorates myocardial damage caused by infectious CLP by reducing the accumulation of superoxide anions caused by sepsis.

The scheme is as follows:

The method comprises the following steps:

DHE staining:

the paraffin embedding and slicing steps are the same as above.

Dyeing DHE: soaking the slices in xylene for 10min, replacing xylene, soaking for 10min again, and soaking for 2min in the order of 100%, 95%, 80% ethanol and deionized water for dewaxing to water for dyeing; after dewaxing, incubating for 30min at 37 ℃ by using DHE staining solution, properly washing, observing and photographing under a fluorescence microscope, wherein tissue sections under the fluorescence microscope show red fluorescence positive staining parts, randomly finding 20-30 non-overlapping visual fields for each section, and semi-quantitatively calculating the relative content of positive substances by adopting medical Image analysis software Image-J5.0 software.

As a result:

as shown in fig. 8, the mice cardiac muscle tissue DHE staining result showed that the fluorescence intensity of the red fluorescence part was significantly increased and significantly increased after CLP injury (P <0.05), and the fluorescence part was significantly decreased and significantly decreased after PSO treatment (P <0.05) compared to the control group.

Claims

Use of PSO for the preparation of a medicament for the treatment and/or prevention of sepsis.
Use of PSO for the preparation of a medicament for the treatment and/or prevention of sepsis-induced myocardial injury.
3. The use of claim 1 or 2, wherein the medicament is an intravenous formulation.
4. The use according to claim 1 or 2, wherein the medicament is administered in a dose of from 12.5mg to 50mg pso per kg body weight.