Application of Liushen pill in preparation of medicine for preventing and treating sepsis

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of Liushen pills in preparation of drugs for preventing and treating sepsis. The treatment effect of the Liushen pill on sepsis is systematically evaluated through a sepsis mouse model induced by a cecum ligation perforation (CLP) method. Research results show that the Liushen pill has remarkable efficacy in improving the survival rate of sepsis mice, inhibiting inflammatory response, relieving oxidative stress, protecting intestinal mucosal barrier function and relieving multi-organ injury, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to the application of Liushen Pills in the preparation of drugs for preventing and treating sepsis. Background Art

[0002] Sepsis is a systemic inflammatory response syndrome (SIRS) triggered by infection, and its pathophysiological process is complex and diverse, involving multiple aspects such as systemic inflammatory response, immune paralysis, oxidative stress, coagulation dysfunction, and multiple organ dysfunction. Its core mechanism is the systemic inflammatory response syndrome (SIRS), and inflammatory factors such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) play key roles in the pathogenesis of sepsis. In addition, sepsis is often accompanied by immune dysfunction, manifested as immune paralysis and immunosuppression, resulting in an increased risk of secondary infection in patients. Oxidative stress and coagulation dysfunction are also important pathophysiological characteristics of sepsis, and they interact with each other, further aggravating tissue damage and organ dysfunction.

[0003] Currently, Western medicine focuses on controlling infection, stabilizing circulation, and supporting organ function. Broad-spectrum antibiotics are used for empirical treatment and adjusted according to etiological results. Glucocorticoids (such as dexamethasone acetate and hydrocortisone) are used for patients with refractory shock. Organ support technologies such as mechanical ventilation and CRRT are indispensable in critical care. Although new therapies such as immunomodulators (tocilizumab) and blood purification technologies are under research, their clinical effects are limited and controversial.

[0004] However, Western medicine treatment has significant drawbacks: Glucocorticoids may exacerbate immunosuppression and increase the risk of secondary infection; the abuse of broad-spectrum antibiotics exacerbates the spread of drug-resistant bacteria, and some drugs (such as vancomycin) have nephrotoxicity; excessive fluid replacement is likely to cause pulmonary edema. These limitations have prompted the academic community to explore safer alternative solutions.

[0005] Traditional Chinese medicine demonstrates unique advantages through "overall regulation" and "treatment based on different stages". Research has confirmed that traditional Chinese medicine ingredients (such as rhubarb and salvia miltiorrhiza) can regulate the inflammatory response through multiple targets, inhibit pro-inflammatory factors such as TNF-α, and at the same time regulate immune homeostasis; Chuanxiong and safflower improve tissue perfusion, while coptis chinensis and rhubarb reduce bacterial translocation by protecting the intestinal barrier. The concept of "treating both the symptoms and the root cause" of traditional Chinese medicine provides an integrated strategy for sepsis from inflammation control to organ repair.

[0006] Chinese Patent Application CN106943541A discloses a traditional Chinese medicine composition for treating sepsis-induced renal dysfunction and its preparation method. The raw materials of its active ingredients are Plantago asiatica, Salvia miltiorrhiza, styrax benzoin, Astragalus membranaceus, artificial bezoar, Scorpio, Rheum officinale, and Imperata cylindrica. The traditional Chinese medicine composition of the present invention has the effects of replenishing qi and promoting blood circulation, detoxifying and promoting diuresis, and purging the bowels and promoting osmosis. It is used for sepsis-induced renal dysfunction, can significantly reduce serum creatinine, urea nitrogen, cystatin, etc., increase urine output, improve renal hemodynamic parameters, inhibit inflammatory factors, and protect endothelium, etc.

[0007] Another Chinese Patent Application CN202410298404.7 discloses a traditional Chinese medicine composition, its preparation method and application for preventing or treating sepsis and target organ damage; the components of the traditional Chinese medicine composition are as follows by weight: 10-15 parts of Rheum officinale, 10-15 parts of Aurantii Fructus Immaturus, 15-25 parts of Magnoliae Officinalis Cortex, 12-23 parts of Angelicae Sinensis Radix, 38-52 parts of Paeoniae Radix Alba, 10-18 parts of Rehmanniae Radix Praeparata, 32-48 parts of Astragalus membranaceus, 10-15 parts of Mori Cortex, 8-17 parts of Glycyrrhizae Radix, 15-25 parts of Ophiopogonis Radix, 10-15 parts of Aster tataricus, 9-16 parts of Hoveniae Semen; the components of the traditional Chinese medicine composition can coordinate and promote each other, and play a role in reducing sepsis and target organ damage through synergistic enhancement, but the composition of this composition is relatively complex.

[0008] Although certain explorations have been made in its treatment in the prior art, its efficacy in comprehensively treating sepsis still needs to be further improved.

[0009] Liushen Pills originated from the prescription of "Leiyunshang Songfentang" pharmacy. It is a famous prescription for clearing heat and detoxifying in the theory of epidemic febrile diseases. The prescription and process of Liushen Pills produced by Leiyunshang Pharmaceutical Group Co., Ltd. are top-secret items, and the specific formula and process are not provided externally. It is mainly prepared from 6 kinds of medicines such as Moschus, Calculus Bovis, Realgar, Venenum Bufonis, etc. It has the effects of clearing heat and detoxifying, and anti-inflammatory and analgesic. It is mainly used for treating acute communicable exanthematous disease with sore throat, swelling and pain of the throat, throat wind and throat carbuncle, single and double tonsillitis, infantile heat boils, carbuncles and sores, breast abscess and back pain, and unknown toxic swelling, etc. It is widely used in multiple departments such as the respiratory department, otolaryngology department, stomatology department, dermatology department, general surgery department, oncology department, etc. There is currently no application of this formula in sepsis. Summary of the Invention

[0010] In view of the deficiencies in the prior art, the present invention provides the application of Liushen Pills in the preparation of drugs for preventing and treating sepsis.

[0011] In order to achieve the object of the present invention, the following technical solutions are adopted:

[0012] The application of Liushen Pills in the preparation of drugs for preventing and treating sepsis.

[0013] Preferably, the Liushen Pills can prevent and treat the systemic inflammation caused by sepsis.

[0014] Preferably, the Liushen Pills can prevent and treat the intestinal tissue oxidative stress reaction complicated by sepsis.

[0015] Preferably, the Liushen Pills can prevent and treat the intestinal mucosal inflammatory reaction and / or barrier function injury caused by sepsis.

[0016] Preferably, the Liushen Pills can prevent and treat the multiple organ dysfunction syndrome complicated by sepsis.

[0017] Preferably, the multiple organ dysfunction syndrome is a syndrome caused by the dysfunction of one or more of the lungs, liver, kidneys, heart, and skeletal muscles.

[0018] Preferably, the Liushen Pills are replaced by the traditional Chinese medicine composition in the Liushen Pills.

[0019] Preferably, the dosage form of the drug is tablets, capsules, granules, powders, liquid preparations, ointments, injections, or spray preparations.

[0020] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0021] Preferably, the traditional Chinese medicine composition is the traditional Chinese medicine extract obtained by extracting each Chinese medicinal material in the Liushen Pills.

[0022] Preferably, the pharmaceutically acceptable excipients include, but are not limited to, binders such as syrup, starch paste, gum arabic, gelatin, tragacanth, or polyvinylpyrrolidone; fillers such as starch, lactose, microcrystalline cellulose, sugar, calcium phosphate, or sorbitol; disintegrants such as sodium carboxymethyl starch, cross-linked sodium carboxymethyl cellulose; lubricants for tableting such as magnesium stearate or talc powder; surfactants such as sodium dodecyl sulfate; suspending agents such as cellulose excipients, gum excipients, aluminum stearate gel, or hydrogenated edible fat; emulsifiers such as lecithin, sorbitan oleate, or gum arabic; preservatives such as methyl paraben or sorbic acid; flavoring agents, coloring agents, and solubilizing agents.

[0023] Preferably, the Liushen Pills are replaced by the Liushen Pills extract prepared from the raw materials of the Liushen Pills.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] (1) In this study, a gold-standard model of sepsis mice induced by cecal ligation and puncture (CLP) method was used to systematically evaluate the therapeutic effect of Liushen Pills on sepsis. The research results show that Liushen Pills exhibit significant pharmacodynamic effects in improving the survival rate of sepsis mice, inhibiting inflammatory reactions, reducing oxidative stress, protecting intestinal mucosal barrier function, and reducing multiple organ damage.

[0026] (2) In Example 1 of the present invention, the high-dose group has a treatment effect comparable to that of the positive drug dexamethasone, and even shows better performance in some indicators. In addition, although a certain treatment effect is shown in some indicators of Comparative Example 1, its overall effect is significantly lower than that of the complete formula of Liushen Pills, indicating that the multi-component synergistic effect of Liushen Pills may be an important mechanism for its efficacy.

[0027] (3) The 7-day survival rates of the high-dose group in Example 1 of the present invention and the positive drug group both reach 90%, which is significantly higher than that of the model group (30%), indicating that Liushen Pills can significantly improve the survival rate of septic mice.

[0028] (4) Each dose group of Liushen Pills can significantly reduce the levels of IL-1β, IL-6 and TNF-α in the serum. Among them, the high-dose group in Example 1 shows the best performance in inhibiting inflammatory factors, which is similar to the positive drug group. As a traditional Chinese medicine compound, the multi-components of Liushen Pills can achieve a more comprehensive anti-inflammatory effect through multi-target synergistic effects, balance the pro-inflammatory and anti-inflammatory responses, and thus reduce the systemic inflammatory response caused by sepsis.

[0029] (5) The SOD activity in the intestinal tissues of the mice in the high-dose group of Example 1 is significantly increased, and the MDA content is decreased, indicating that it has strong antioxidant capacity, which may be related to various natural antioxidant substances in its components. For example, components such as moschus and calculus bovis have the effects of scavenging free radicals and protecting cell membranes. This antioxidant capacity helps to reduce tissue damage caused by sepsis, especially in vulnerable organs such as the intestine. The serum D-lactic acid level of the septic mice in the high-dose group of Example 1 is significantly decreased, and the expressions of TNF-α and IL-6 in the ileum tissue are reduced, indicating that it can protect the intestinal mucosal barrier function and reduce the intestinal mucosal inflammatory response. This protective effect may be closely related to its anti-inflammatory and antioxidant effects.

[0030] (6) Liushen Pills significantly reduce the MPO level in the lung tissues of septic mice, as well as the levels of ALT, AST and CRE in the serum, indicating that it has a protective effect on organs such as the lung, liver, heart, skeletal muscle and kidney. This multi-organ protective effect of Liushen Pills may be related to its comprehensive anti-inflammatory and antioxidant capacity. By inhibiting the overexpression of inflammatory factors and reducing oxidative stress, it maintains the integrity of the intestinal mucosa, reduces the translocation of bacteria and toxins, and thus reduces the systemic inflammatory response and multiple organ dysfunction. Detailed implementation manners

[0031] The following further describes the present invention in conjunction with the detailed implementation manners.

[0032] The raw materials, equipment and reagents used in the detailed implementation manners of the present invention are all commercially available conventional products.

[0033] Example 1

[0034] The drug of the present invention: Liushen Pills, a product of Leiyunshang Pharmaceutical Group Co., Ltd., with national medicine standard Z32020481.

[0035] Comparative Example 1

[0036] The disclosed four-flavor medicinal composition: its medicinal materials are composed of musk, bezoar, toad venom and realgar in an amount equal to that of Liushen Pills, and its preparation method is the same as that of Liushen Pills in Example 1, and it is made by Lei Yunshang Pharmaceutical Group Co., Ltd.

[0037] Study on the efficacy of Liushen Pills in treating sepsis in Example 1 of the present invention

[0038] 1. Materials

[0039] 1.1 Animals

[0040] 250 healthy male C57BL / 6J mice, 7 weeks old, were purchased from Shanghai Slake Laboratory Animal Co., Ltd. Animal production license number: SCXK (Shanghai) 2022-0004; Animal use license number: SYXK (Shanghai) 2020-0009. The experimental animals were housed in the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine, with a temperature of (20.0±2.0)℃, a humidity of 40%-70%, and a light / dark cycle of 12h each.

[0041] 1.2 Reagents

[0042] Mouse interleukin-1β (I-1B) ELISA kit was purchased from Wuhan Elaruite Biotechnology Co., Ltd., catalog number: E-EL-M0037;

[0043] Mouse interleukin-6 (IL-6) ELISA kit was purchased from Wuhan Elaruite Biotechnology Co., Ltd., catalog number: E-EL-M0044;

[0044] Mouse tumor necrosis factor-α (TNF-α) ELISA kit was purchased from Wuhan Elaruite Biotechnology Co., Ltd., catalog number: E-EL-M3063;

[0045] The total superoxide dismutase (T-SOD) assay kit was purchased from Nanjing Jiancheng Bioengineering Research Institute, catalog number: A001-3-2;

[0046] Malondialdehyde (MDA) assay kit was purchased from Nanjing Jiancheng Bioengineering Research Institute, catalog number: A003-1-2;

[0047] The serum D-lactic acid kit was purchased from BioVision, USA, catalog number K667-100;

[0048] Myeloperoxidase (MPO) kit was purchased from Nanjing Jiancheng Bioengineering Institute, catalog number A044-1-1;

[0049] Alanine aminotransferase (ALT) kit was purchased from Nanjing Jiancheng Bioengineering Institute, catalog number C009-1-1;

[0050] Aspartate aminotransferase (AST) kit was purchased from Nanjing Jiancheng Bioengineering Institute, catalog number C010-2-1;

[0051] Mouse creatinine (Cr) ELISA kit was purchased from Shanghai ELISA Biotechnology Co., Ltd., catalog number ml037726;

[0052] Ketamine hydrochloride injection was purchased from Fujian Gutian Pharmaceutical Co., Ltd., approval number: National Medicine Standard H35020148.

[0053] 2. Experimental Methods

[0054] 2.1 Animal grouping and modeling

[0055] 250 healthy male C57BL / 6J mice were randomly selected as sham operation group after 1 week of adaptive feeding, and 215 mice were randomly selected to induce sepsis model by cecal ligation and puncture (CLP): mice were fasted for 8 hours before operation, anesthetized by isoflurane inhalation, and fixed after anesthesia. The abdominal hair of mice was shaved, and the abdominal skin was disinfected with 75% alcohol. Then, 1-2 cm was cut along the midline of the abdomen, and the epidermis and muscle layer were separated to expose the cecum. The cecal contents were squeezed to the end of the cecum, and the cecum was ligated with 4-0 surgical absorbable moderate sepsis ligature suture at 3 / 4 of the end. After ligation, a 1.2×38TWLB needle was used to puncture the cecal wall at the blind end of the cecum. After squeezing out a small amount of intestinal contents, the cecum was returned to avoid contamination. The muscle layer and skin were sutured layer by layer. After surgery, each mouse was subcutaneously injected with 1 mL of 37℃ preheated saline for fluid replacement. After warming and resuscitation, the mice were placed in cages for observation. After modeling, they were randomly divided into 6 groups: model group, low-dose group in Example 1, medium-dose group in Example 1, high-dose group in Example 1, comparative example 1 group, and positive drug group, with 35 mice in each group. The 35 mice in the sham operation group were not subjected to cecal ligation and puncture, and the rest of the operations were the same as the CLP method.

[0056] 2.2 Drug preparation and administration method

[0057] 2.2.1 Drug preparation

[0058] (1) Preparation of drugs for the low, medium, and high-dose groups in Example 1: Weigh the Liushen Pills in Example 1, crush them, add pure water, and obtain a suspension by ultrasonic treatment. Prepare a suspension with a concentration of 5 mg / ml for animal administration in the low, medium, and high-dose groups of Liushen Pills, and use it immediately after preparation.

[0059] (2) Preparation of drugs for the control group in Comparative Example 1: Weigh the same amount of moschus, bezoar, bufotoxin, and realgar as in the high-dose group of Example 1, add pure water, and prepare a suspension with a concentration of 5 mg / ml for animal administration in the control group of Comparative Example 1, and use it immediately after preparation.

[0060] (3) Positive drug group: Dexamethasone sodium phosphate injection, purchased from Chenxin Pharmaceutical Co., Ltd. (batch number H37021969).

[0061] 2.2.2 Administration method

[0062] The sham operation group and the model group were gavaged with pure water (0.2 mL) once a day; the low-dose group, medium-dose group, high-dose group of Example 1, and the control group of Comparative Example 1 were gavaged with the corresponding drug suspension once a day; the positive drug group was intraperitoneally injected with dexamethasone sodium phosphate injection (0.2 mL) once a day, and the first administration was performed 2 hours after modeling. The specific grouping and administration are shown in Table 1.

[0063] Table 1 Animal grouping and administration table

[0064]

[0065]

[0066] 2.2.3 Sample collection

[0067] After numbering the mice in each group, 10 were randomly selected for status observation; for the remaining 25, 6 surviving mice were randomly selected 24 hours after modeling, anesthetized by intraperitoneal injection of ketamine at a dose of 1.0 - 1.5 mg / 10 g, then blood was collected from the abdominal aorta, and small intestine tissues were taken and stored at low temperature; 6 mice in each group were taken 72 hours after modeling, anesthetized by intraperitoneal injection of ketamine at a dose of 1.0 - 1.5 mg / 10 g, then blood was collected from the abdominal aorta, and lung tissues were taken and stored at low temperature.

[0068] 3. Experimental results

[0069] 3.1 Mouse status

[0070] Sepsis is a disease with a high mortality rate, and the improvement of survival rate can directly reflect the therapeutic effect of drugs on sepsis.

[0071] Ten mice were randomly selected from each group and observed every 12 h. The status within 7 days after surgery was recorded, and the survival rates at 1, 3, and 7 days of each group were calculated. Log-rank test was performed using Graphpad 8 to calculate the P value of the survival rate on the 7th day between groups and draw the survival curve. The results are shown in Table 2.

[0072] Table 2 Effects of Liushen Pills on the survival rate of septic mice

[0073] Group 1-day survival rate (%) 3-day survival rate (%) 7-day survival rate (%) Sham operation group 100 100 <![CDATA[100 ** > Model group 70 40 30 Low-dose group of Example 1 90 60 40 Medium-dose group of Example 1 80 70 60 High-dose group of Example 1 90 90 <![CDATA[90 ## > Group of Comparative Example 1 90 70 60 Positive drug group 100 90 <![CDATA[90 ## >

[0074] Note: Compared with the sham operation group, *P < 0.05, **P < 0.01; compared with the model group, #P < 0.05, ##P < 0.01; compared with the high-dose group of Example 1, &P < 0.05, &&P < 0.01.

[0075] The hair of the mice in the sham operation group was shiny, lively and active, and the diet was normal. All survived after 7 d. The hair of the mice in the model group was dull, and the diet, drinking water and daily activity intensity decreased significantly. Three mice died within 24 h. The survival rates at 24 h, 72 h and 7 d were 70%, 40% and 30% respectively. The hair, diet and activity status of the mice in the low-dose group of Example 1, the medium-dose group of Example 1, the high-dose group of Example 1, the control group 1 and the positive drug group were improved to varying degrees compared with the model group. Among them, the high-dose group of Example 1 and the positive drug group were the best, and the status was similar to that of the sham operation group. The survival rate at 7 d was 90%, which was significantly higher than that of the model group (P < 0.01). The survival rate of the control group 1 was significantly lower than that of the high-dose group of Example 1 (P < 0.01).

[0076] 3.2 Inflammatory response

[0077] Sepsis is a systemic inflammatory response syndrome. Inflammatory factors such as IL-1β, IL-6, and TNF-α play important roles in the pathological process of sepsis. The increase in the levels of these inflammatory factors will lead to systemic inflammatory response and multiple organ dysfunction. The changes in the levels of inflammatory factors can reflect the anti-inflammatory effect of drugs and their intervention on the pathological process of sepsis.

[0078] At 24 h after modeling, six mice from each group were anesthetized by intraperitoneal injection of 1.0 - 1.5 mg / 10 g ketamine, and blood was collected from the abdominal aorta. After the blood specimens were allowed to stand and centrifuged, the serum was taken, and the levels of IL-1β, IL-6, and TNF-α in the mouse serum were detected by ELISA method. The operation was carried out strictly according to the instructions of the kit. The results are shown in Table 3.

[0079] Table 3 Effects of Liushen Pills on the levels of IL-1β, IL-6 and TNF-α in the serum of septic mice (n = 6)

[0080] Group IL-1β (pg / ml) IL-6 (pg / ml) TNF-α (pg / ml) Sham operation group 24.67±1.26 75.51±3.95 85.55±5.93 Model group <![CDATA[66.40±3.01 ** > <![CDATA[231.57±12.17 ** > <![CDATA[296.32±11.38 ** > Low-dose group of Example 1 <![CDATA[62.19±1.71 # > 205.48±91.29 <![CDATA[272.50±84.98 # > Medium-dose group of Example 1 <![CDATA[48.45±0.92 ## > <![CDATA[98.71±8.83 ## > <![CDATA[179.71±7.88 ## > High-dose group of Example 1 <![CDATA[34.09±0.93 ## > <![CDATA[60.44±8.93 ## > <![CDATA[149.02±12.81 ## > Group of Comparative Example 1 <![CDATA[35.46±1.70 ## > <![CDATA[124.17±14.32 ##&& > <![CDATA[170.43±19.44 ##& > Positive drug group <![CDATA[35.53±1.07 ##& > <![CDATA[105.03±16.86 ##&& > <![CDATA[159.12±9.20 ## >

[0081] Note: Compared with the sham operation group, *P < 0.05, **P < 0.01; compared with the model group, #P < 0.05, ##P < 0.01; compared with the high-dose group of Example 1, &P < 0.05, &&P < 0.01.

[0082] The levels of IL-1β, IL-6 and TNF-α in the serum of mice in the model group were significantly higher than those in the sham operation group (P < 0.01); compared with the model group, the levels of IL-1β, IL-6 and TNF-α in the serum of mice in the low-dose group of Example 1 decreased (P < 0.05), and the levels of IL-1β, IL-6 and TNF-α in the serum of mice in the medium-dose group of Example 1, the high-dose group of Example 1, the control group 1 and the positive drug group decreased significantly (P < 0.01); compared with the control group 1, the levels of IL-6 and TNF-α in the serum of mice in the high-dose group of Example 1 decreased significantly (P < 0.01); the overall levels of inflammatory factors in the high-dose group of Example 1 were similar to those in the positive drug group (P > 0.05).

[0083] The above results indicate that Liushen Pills and its combination of four herbs have a significant inhibitory effect on the systemic inflammatory response caused by sepsis, and the effect of the complete formula of Liushen Pills is better than that of its combination of four herbs.

[0084] 3.3 Oxidative stress in intestinal tissue

[0085] Sepsis patients are often accompanied by oxidative stress reactions. Superoxide dismutase (SOD) is an important antioxidant enzyme in the body, and malondialdehyde (MDA) is a product of lipid peroxidation. Both are important indicators reflecting the state of oxidative stress.

[0086] At 24 h after modeling, 6 mice in each group were anesthetized by intraperitoneal injection of 1.0 - 1.5 mg / 10 g ketamine, and small intestine tissues were ground in an ice bath to prepare tissue homogenates. The supernatant was taken by centrifugation, and the SOD activity and MDA content in the intestinal tissues were detected by the xanthine oxidase method and the thiobarbituric acid colorimetric method respectively, and the operation was carried out strictly according to the instructions of the kit. The results are shown in Table 4.

[0087] Table 4 Effects of Liushen Pills on the content of MAD and SOD activity in the small intestine tissues of septic mice (n = 6)

[0088] Group MDA (nmol / L) SOD (U / mg) Sham operation group 7.16±0.47 71.71±5.61 Model group <![CDATA[25.71±2.75 ** > <![CDATA[35.78±3.47 ** > Low-dose group of Example 1 24.15±1.48 31.01±4.09 Medium-dose group of Example 1 <![CDATA[17.80±1.37 ## > <![CDATA[61.14±3.04 ## > High-dose group of Example 1 <![CDATA[9.58±0.86 ## > <![CDATA[76.32±5.19 ## > Group of Comparative Example 1 <![CDATA[21.51±1.52 ##&& > <![CDATA[33.33±3.48 && > Positive drug group <![CDATA[10.88±1.13 ##& > <![CDATA[84.20±5.77 ##& >

[0089] Note: Compared with the sham operation group, *P < 0.05, **P < 0.01; compared with the model group, #P < 0.05, ##P < 0.01; compared with the high-dose group of Example 1, &P < 0.05, &&P < 0.01.

[0090] Compared with the sham operation group, the content of MAD in the small intestine tissue of mice in the model group was significantly increased, and the activity of SOD was decreased (P < 0.01); while the content of MAD and the activity of SOD in the small intestine tissue of mice in the low-dose group of Example 1 were comparable to those in the model group (P > 0.05); the content of MAD in the small intestine tissue of mice in the medium-dose group of Example 1, the high-dose group of Example 1, and the positive drug group was significantly decreased, and the activity of SOD was increased (P < 0.01), and the high-dose group of Example 1 was slightly better than the positive drug group; the content of MAD in the small intestine tissue of mice in the control group 1 was decreased (P < 0.01), and the activity of SOD was similar (P > 0.05), showing a significant difference from the high-dose group of Example 1 (P < 0.05).

[0091] It shows that the complete formula of Liushen Pills can significantly improve the oxidative stress response of the body caused by sepsis at high doses, has antioxidant ability, can protect related tissues, and weaken the damage caused by sepsis.

[0092] 3.4 Intestinal mucosal permeability

[0093] The increase in intestinal mucosal permeability is one of the important characteristics in the pathological process of sepsis. The destruction of the intestinal mucosal barrier function will lead to the translocation of bacteria and toxins, further aggravating the systemic inflammatory response and multiple organ dysfunction. D-lactic acid is a product of intestinal bacteria metabolism. Under normal circumstances, when the intestinal mucosal barrier is intact, D-lactic acid is difficult to enter the blood circulation through the intestinal mucosa. In sepsis, the intestinal mucosal barrier function is damaged and the permeability increases, resulting in D-lactic acid entering the blood circulation from the intestine, and the serum D-lactic acid level increases. The increase in serum D-lactic acid level is a direct marker of increased intestinal mucosal permeability and can intuitively reflect the degree of damage to the intestinal mucosal barrier function. Tumor necrosis factor-α (TNF-α) is an important pro-inflammatory factor. In sepsis, the overexpression of TNF-α will lead to intestinal mucosal inflammatory response and barrier function damage.

[0094] At 72 h after modeling, 6 mice in each group were anesthetized by intraperitoneal injection of ketamine at 1.0 - 1.5 mg / 10 g, and blood was collected from the abdominal aorta. After the blood samples were allowed to stand and centrifuged, the serum was taken, and the serum D-lactic acid level of mice was detected by ELISA method; the ileum tissue was ground in an ice bath to make a tissue homogenate, and the supernatant was taken after centrifugation, and the TNF-α and IL-6 levels in the ileum tissue of mice were detected by ELISA method, and the operation was carried out strictly according to the kit instructions. The results are shown in Table 5.

[0095] Table 5 Effects of Liushen Pills on serum D-lactic acid, TNF-α and IL-6 levels in ileum tissue of septic mice (n = 6)

[0096] Group D-lactic acid (μmol / L) TNF-α (pg / mL) IL-6 (pg / mL) Sham operation group 171.78±12.94 38.86±1.76 83.17±9.45 Model group <![CDATA[538.89±74.98 ** > <![CDATA[200.05±55.44 ** > <![CDATA[805.06±118.35 ** > Low-dose group of Example 1 466.71±80.02 183.81±51.79 <![CDATA[614.47±111.37 # > Medium-dose group of Example 1 <![CDATA[393.54±46.21 ** > <![CDATA[122.05±41.45 ## > <![CDATA[654.74±112.17 # > High-dose group of Example 1 <![CDATA[335.94±69.57 ## > <![CDATA[58.77±9.63 ## > <![CDATA[258.16±38.65 ## > Group of Comparative Example 1 <![CDATA[424.63±56.17 #& > <![CDATA[209.19±9.44 ##&& > <![CDATA[530.26±104.88 ##&& > Positive drug group <![CDATA[274.23±25.40 ## > <![CDATA[38.68±12.29 ##& > <![CDATA[264.08±40.22 ## >

[0097] Note: Compared with the sham operation group, *P < 0.05, **P < 0.01; compared with the model group, #P < 0.05, ##P < 0.01; compared with the high-dose group of Example 1, &P < 0.05, &&P < 0.01.

[0098] The levels of D-lactic acid in the serum of mice in the model group, TNF-α and IL-6 in the ileum tissue were significantly higher than those in the sham operation group (P < 0.01). The level of D-lactic acid in the serum of mice in the low-dose group of Example 1 was slightly lower than that in the model group, but the difference was not significant (P > 0.05), and the level of IL-6 decreased significantly (P < 0.05). The levels of D-lactic acid in the serum and TNF-α in the ileum tissue of mice in the medium-dose group of Example 1 were significantly lower than those in the model group (P < 0.01); the levels of D-lactic acid in the serum, TNF-α and IL-6 in the ileum tissue of mice in the high-dose group of Example 1 and the positive drug group were significantly lower than those in the model group (P < 0.01), and the levels of D-lactic acid and IL-6 in the serum of the two groups of mice were similar (P > 0.05). The levels of D-lactic acid in the serum, TNF-α and IL-6 in the ileum tissue of the control group 1 were lower than those in the model group (P < 0.05), but were significantly higher than those in the high-dose group of Example 1 (P < 0.01).

[0099] It shows that the complete formula of Liushen Pills can weaken the damage of sepsis to the intestinal mucosal barrier function and inhibit the intestinal mucosal inflammatory response at high doses, and the medium dose and the combination of four herbs also have therapeutic effects on the intestinal mucosal injury caused by sepsis.

[0100] 3.5 Multiple organ injuries

[0101] In the study of sepsis, multiple organ dysfunction syndrome (MODS) is one of the main causes of patient death. The systemic inflammatory response and oxidative stress caused by sepsis can lead to functional damage of multiple organs (such as the lungs, liver, kidneys, etc.). Therefore, evaluating multiple organ injuries is of great significance for studying the effect of drugs in treating sepsis.

[0102] The increase in the level of MPO in the lung tissue directly reflects the degree of neutrophil-mediated inflammatory response and oxidative stress, and is an important indicator for evaluating sepsis-related acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). The increase in the level of serum ALT is a sensitive indicator of hepatocyte injury and reflects the damage of sepsis to liver function. The increase in the level of serum AST reflects the damage of multiple organs such as the liver, heart, and skeletal muscle, and is an important indicator for evaluating sepsis-related multiple organ dysfunction. The increase in the level of serum CRE directly reflects the degree of renal function damage. By monitoring the change in the level of serum CRE, the intervention effect of drugs on sepsis-related kidney injury can be evaluated, and the protective effect of drugs on kidney function can be reflected.

[0103] At 72 h after modeling, 6 mice in each group were anesthetized by intraperitoneal injection of ketamine at a dose of 1.0 - 1.5 mg / 10 g. Lung tissues were taken and ground into tissue homogenates in an ice bath. After centrifugation, the supernatant was taken, and the MPO level in the lung tissues of mice was detected using a kit. Blood was collected from the abdominal aorta. After the blood samples were allowed to stand and then centrifuged, the serum was taken, and the expression levels of ALT, AST, and CRE in the serum of mice were detected using a kit. The operations were carried out strictly according to the kit instructions. The results are shown in Table 6.

[0104] Table 6 Effects of Liushen Pills on the levels of MPO in lung tissues, and ALT, AST, and CRE in serum of septic mice (n = 6)

[0105]

[0106] Note: Compared with the sham operation group, *P < 0.05, **P < 0.01; compared with the model group, #P < 0.05, ##P < 0.01; compared with the high-dose group of Example 1, &P < 0.05, &&P < 0.01.

[0107] The levels of MPO in the lung tissues and ALT, AST, and CRE in the serum of mice in the model group were significantly higher than those in the sham operation group (P < 0.01). The MPO level in the lung tissues of mice in each dose group of the examples and the group of Comparative Example 1 decreased significantly compared with the model group (P < 0.01). However, there was no significant difference in the levels of ALT, AST, and CRE in the serum of mice in the low-dose group of Example 1 and the model group (P > 0.05). The levels of ALT, AST, and CRE in the serum of mice in the medium- and high-dose groups of the examples decreased significantly compared with the model group (P < 0.01); the data of the high-dose group of Example 1 were similar to those of the positive drug group (P > 0.05) and were significantly lower than those of the group of Comparative Example 1 (P < 0.01).

[0108] It shows that the complete formula of Liushen Pills and the combination of four herbs both have anti-inflammatory and antioxidant effects, and can weaken the pulmonary inflammation and oxidative stress response caused by sepsis. The complete formula of Liushen Pills can weaken the damage to hepatocytes, cardiomyocytes, skeletal muscle cells, and kidneys caused by sepsis and has a protective effect on multiple organ functions at medium and high doses.

[0109] The above detailed description is a specific description of one feasible embodiment of the present invention. This embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification without departing from the present invention should be included within the scope of the technical solution of the present invention.

Claims

1. Application of Liushen Pills in the preparation of drugs for preventing and treating sepsis.

2. The application according to claim 1, characterized in that The Liushen Pills can prevent and treat one or more of systemic inflammation, intestinal tissue oxidative stress response, intestinal mucosal inflammatory response and / or barrier function injury, and multiple organ dysfunction syndrome caused by sepsis.

3. The application according to claim 1, wherein The Liushen Pills can prevent and treat systemic inflammation caused by sepsis.

4. The application according to claim 1, characterized in that The Liushen Pills can prevent and treat intestinal tissue oxidative stress response complicated by sepsis.

5. The application according to claim 1, characterized in that, The Liushen Pills can prevent and treat intestinal mucosal inflammatory response and / or barrier function injury caused by sepsis.

6. The application according to claim 1, characterized in that, The Liushen Pills can prevent and treat multiple organ dysfunction syndrome complicated by sepsis.

7. The application according to claim 6, characterized in that, The multiple organ dysfunction syndrome is a syndrome caused by functional injury of one or more of the lungs, liver, kidneys, heart, and skeletal muscles.

8. The application according to any one of claims 1-6, characterized in that, The raw materials of Liushen Pills are prepared into tablets, capsules, granules, powders, liquid preparations, ointments, injections or spray preparations.

9. The application according to claim 8, wherein The drug also includes pharmaceutically acceptable excipients.

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