Application of rhizoma coptidis-fructus gardeniae medicine pair in preparation of anti-sepsis medicine and anti-sepsis metabonomics research of rhizoma coptidis-fructus gardeniae medicine pair

Abstract

The invention discloses application of a rhizoma coptidis-fructus gardeniae medicine pair in preparation of a medicine for resisting sepsis, a sepsis model is constructed through CLP in an experiment, and after administration intervention of the rhizoma coptidis-fructus gardeniae medicine pair, the death rate of mice with sepsis can be remarkably reduced, and the inflammation and infection conditions of the mice can be reduced. In addition, small molecule metabolites in serum of sepsis mice are analyzed by using a metabonomics technology. Research finds that metabolic pathways such as amino acid metabolism play a key role in the serum metabolism regulation process of the coptis chinensis-gardenia medicine for resisting sepsis, the potential target and mechanism of the medicine for intervening sepsis metabolic disorder are disclosed, and a reference basis is provided for preventing and treating sepsis and screening other medicines for treating sepsis.

Description

TECHNICAL FIELD

[0001] The application relates to a quality control method of traditional Chinese medicine, in particular to a quality detection method of a Huanglian-Zhizi medicinal pair. BACKGROUND

[0002] The medicinal pair is a commonly used compatibility form in clinical medicine prescription of traditional Chinese medicine, is an experience summary of clinical medicine use by medicine scholars in generations, and is used in combination with Huanglian and Zhizi, is derived from Huanglian Jiedu Decoction (HLJDD) in Wai Tai Mi Yao, has the effects of clearing the intestines and purging fire, and is clinically used for treating enteritis, esophagitis, and aphtha.

[0003] Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection, and is an inflammatory response throughout the body caused by invasion of pathogens, and the imbalance between infectious pathogens and anti-inflammatory response is the main feature of sepsis. The cecal ligation and puncture (CLP) operation is the gold standard for constructing a sepsis animal model. The pathogenesis of 'toxic heat' is the key to the onset of sepsis. However, the pathogenesis and fatal causes of sepsis have not been completely elucidated. Sepsis is usually caused by a dysregulated immune response triggered by systemic infection, and the pathological process of sepsis is extremely complex. Traditional Chinese medicine can improve the metabolic state of the body due to its overall regulation characteristics, and has unique advantages in the prevention and treatment of diseases. Since the metabolic process of traditional Chinese medicine in the body presents dynamic changes, which is highly consistent with the concept of 'holism, dynamics, comprehensiveness and analysis' emphasized by metabolomics, metabolomics technology has a significant advantage in analyzing the metabolic rules of traditional Chinese medicine. Applying it to the research and development of traditional Chinese medicine can not only help to reveal the material basis and mechanism of action of effective components of traditional Chinese medicine, but also provide a scientific basis for precise medication dosage in clinical practice. In recent years, metabolomics has been widely used in the study of sepsis and related diseases.

[0004] Previous studies have shown that the efficacy of the Huanglian-Zhizi combination is significantly better than that of single-ingredient medicine, and its preventive administration can significantly reduce the mortality rate of sepsis mice, reduce the levels of infection and inflammatory factors in serum, and effectively alleviate the damage to liver and lung tissues, indicating that the combination has a significant protective effect on sepsis mice. However, the specific molecular mechanism of its prevention of sepsis has not been completely elucidated. SUMMARY

[0005] The purpose of the present application is to solve the problems of the prior art, to construct a sepsis model by CLP operation, to further explore the effect of the Huanglian-Zhizi medicinal pair on sepsis, to lay a foundation for the prevention of sepsis and to provide data support. And by using metabolomics technology, the mechanism of the Huanglian-Zhizi medicinal pair with two different proportions in resisting sepsis is further explored, aiming to provide new ideas and strategies for the prevention and treatment of sepsis.

[0006] Technical scheme: In order to achieve the above purpose, the technical scheme adopted by the present application is:

[0007] Use of a Huanglian-Zhizi pair in the preparation of an anti-sepsis drug.

[0008] As a preferred solution, the use of a Huanglian-Zhizi pair with a mass ratio of 5:5 to 5:9 in the preparation of an anti-sepsis drug.

[0009] As a preferred solution, the extraction method of Huanglian and Zhizi is as follows: Huanglian and Zhizi decoction pieces are taken in a mass ratio, respectively, and soaked in 8 to 16 times the amount of water for 0.5 to 2 hours, and then extracted by reflux for 1 to 3 times, each time for 0.5 to 1.5 hours, filtered, and then the filtrates are combined, concentrated, and freeze-dried to obtain the extract of the Huanglian-Zhizi pair.

[0010] As a preferred solution, the extraction method of Huanglian and Zhizi is as follows: Huanglian and Zhizi decoction pieces are taken in a mass ratio, respectively, and soaked in 8 to 12 times the amount of water for 0.5 to 1 hour, and then extracted by reflux for 2 to 3 times, each time for 0.5 to 1.5 hours, filtered through five layers of gauze, and then the filtrates are combined, concentrated, and freeze-dried to obtain the extract of the Huanglian-Zhizi pair.

[0011] The present application provides a metabonomics research method for the anti-sepsis effect of a Huanglian-Zhizi pair, which comprises the following steps:

[0012] (1) Animal grouping and administration

[0013] After adaptive feeding for one week, the mice were randomly divided into 8 groups, namely, a sham operation group, a model group, a Huanglian LH group, a Huanglian HH group, a Zhizi ZZ group, a Huanglian-Zhizi pair with a mass ratio of 5:9 group, a Huanglian-Zhizi pair with a mass ratio of 9:9 group, a Xuebijing group, and a Xuebijing group. The mice in the Xuebijing group were administered intraperitoneally, and the mice in the other groups were administered orally, for 7 consecutive days. The mice in the sham operation group and the model group were given pure water in a corresponding volume. Two hours after the last administration, a cecal ligation and puncture operation was performed to model the mice. Twenty-four hours after modeling, the mice were enucleated to collect blood, and the serum samples were stored in a -80℃ refrigerator. Immediately after blood collection, the mice were executed by cervical dislocation, and the liver, lung tissue, and cecal contents were dissected for subsequent detection.

[0014] (2) Serum sample processing method

[0015] After being taken out of the -80℃ refrigerator, the serum samples were thawed at 4℃, and then methanol was added thereto. After vortexing and centrifugation, the supernatant was removed, vacuum centrifuged to dryness, and then redissolved in a cold acetonitrile aqueous solution. After vortexing and centrifugation again, the supernatant was obtained for analysis.

[0016] (3) Metabonomics analysis

[0017] The serum samples of each group in step (2) were subjected to UPLC-Q-TOF / M metabonomics analysis.

[0018] (4) Data preprocessing

[0019] Progenesis QI: The raw data of Waters instrument was imported into Progenesis QI software for peak alignment, peak extraction, peak identification and normalization processing, and EZinfo 3.0 software was used for data analysis;

[0020] MS-DIAL: The raw data of Waters instrument was imported into MSConvertGUI software, and converted into.mzML format file; then, these files were imported into MS-DIAL ver 4.24, and subsequent operations were performed through data collection, peak extraction, identification, normalization steps; combined with the results of the two software, the differential metabolites with VIP>1 and p<0.05 were selected for subsequent analysis;

[0021] (5) Identification and pathway analysis of differential metabolites

[0022] Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used for data analysis, and metabolites with VIP>1 and p<0.05 were selected as potential biomarkers; accurate m / z and retention time were obtained by UPLC-Q-TOF / MS, and corresponding databases Progenesis QI, MSDIAL, HMDB were searched for mass spectrum information matching; potential biomarkers were preliminarily identified according to actual fragment ions and theoretical fragment ions, and the identified markers were imported into MetaboAnalyst (http: / / www.metaboanalyst.ca) for enrichment and analysis of metabolic pathways; Graphpad Prism 8.3.0 software was used for statistical analysis.

[0023] As a preferred scheme, step (1) animal grouping and administration scheme: after one week of adaptive feeding, the mice were randomly divided into 8 groups, namely, sham operation group; model group; Huanglian LH group, dose 0.65 g / kg; Huanglian HH group, dose 1.17 g / kg; Zhizi ZZ group, dose 1.17 g / kg; Huanglian-Zhizi mass ratio 5:9 group, dose 1.82 g / kg; Huanglian-Zhizi mass ratio 9:9 group, dose 2.34 g / kg; Xuebijing group, 10 mL / kg; Xuebijing group mice were intraperitoneally injected, and the rest of the mice were given oral administration, for 7 consecutive days; the sham operation group and the model group mice were given pure water of corresponding volume; 2 h after the last administration, the cecal ligation and puncture operation modeling was performed; 24 h after modeling, the eyeball was removed and blood was taken, and the serum samples were stored at-80°C refrigerator; immediately after blood collection, the mice were executed by cervical dislocation, dissected, and the liver, lung tissue and cecal contents were taken for subsequent detection.

[0024] As a preferred solution, the serum sample processing method of step (2) is as follows: after the serum sample is taken out from a-80 DEG C refrigerator, the serum sample is thawed at 4 DEG C, 50 muL of serum is added into 300 muL of pre-cooled methanol, vortexed for 1 min, centrifuged at 4 DEG C and 12000 rpm for 15 min, 200 muL of supernatant is removed, vacuum centrifuged to dryness, 200 muL of 10% cold acetonitrile aqueous solution is added for re-dissolution, vortexed and centrifuged again, and the supernatant sample is obtained for analysis.

[0025] As a preferred solution, the chromatographic condition of the metabolomics analysis of step (3) is as follows: an ACQUITY UPLC HSS C18 column with a size of 2.1 mm*100 mm, 1.8 mu m, a mobile phase of 0.1% formic acid aqueous solution as phase A-acetonitrile as phase B, a flow rate of 0.30 mL / min, a column temperature of 35 DEG C, and a sample injection amount of 3 muL; and the elution condition is as follows: 0-3.0 min 95%-85% A, 3-6 min 85%-78% A, 6-13 min 78%-5% A, 13-16 min 5%-0% A, 16-17 min 0% A, 17-18 min 0%-95% A, 18-20 min 95% A.

[0026] The mass spectrometry condition is as follows: in a positive ion mode, the capillary voltage is 3.0 KV, the cone hole voltage is 55 V, the desolvation gas temperature is 350 DEG C, the desolvation gas flow is 800 L / h, the ion source temperature is 100 DEG C, and the cone hole gas flow is 50 L / h.

[0027] Advantages: compared with the prior art, the present application has the following advantages:

[0028] In the experiment of the present application, the CLP is used to construct a sepsis model, and after the intervention of the Huanglian-Jizisheng pair, the mortality of the sepsis mice can be significantly reduced, the inflammation and infection of the mice can be reduced, and it is found that the Huanglian-Jizisheng pair can improve the damage of the liver and lung tissues, and the results show that the effect of the combination of Huanglian and Jizisheng is stronger than that of the single medicine, and when the ratio of Huanglian to Jizisheng is 5:9, the effect is more obvious than that of the ratio of 9:9, which shows that the combination of Huanglian and Jizisheng has a synergistic effect.

[0029] The application applies metabolomics technology to analyze small molecule metabolites in serum of sepsis mice. Research finds that, compared with the sham operation group, the content of differential metabolites in serum of sepsis mice presents significant changes, and after intervention of the Huanglian-Zhizi drug pair, phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, alanine, aspartate and glutamate metabolism, arginine and proline metabolism, glycerophospholipid metabolism, primary bile acid biosynthesis, steroid hormone biosynthesis and steroid biosynthesis are significantly regulated. These metabolic pathways play a key role in the serum metabolic regulation process of Huanglian-Zhizi drug pair against sepsis, reveal the potential target and mechanism of the drug for intervention of sepsis metabolic disorder, and provide a reference for prevention and treatment of sepsis and screening of other drugs for treating sepsis. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The survival rate of each group of mice for 24h.

[0031] Figure 2 The effect of Huanglian-Zhizi compatibility on the levels of IL-6, TNF-α, ET-1, PCT, CRP, AST and ALT in serum of sepsis mice.

[0032] Figure 3 The effect of Huanglian-Zhizi compatibility on the pathological changes of liver tissue of sepsis mice.

[0033] Figure 4 The effect of Huanglian-Zhizi compatibility on the pathological changes of lung tissue of sepsis mice.

[0034] Figure 5 The representative BPI chromatogram of serum samples of the Sham group (A) and the CLP group (B) under positive ion mode.

[0035] Figure 6 The PCA, OPLS-DA and PLS-DA diagrams of serum of each group.

[0036] Figure 7 The heat map of 17 common differential metabolites identified by comparison among the Sham group, the CLP group, the LD group and the HD group.

[0037] Figure 8The metabolism pathway map of the combined Huanglian-Rhizoma Japonicae serum against sepsis. DETAILED DESCRIPTION

[0038] The application will be further clarified by the following examples, which should not be construed as limiting the scope of the application. After reading the application, various modifications to the application in addition to those described herein will become apparent to those of ordinary skill in the art from this disclosure. The scope of the application should be determined by reference to the appended claims.

[0039] Example 1 Comparative effect evaluation of the protective effect of Huanglian-Rhizoma Japonicae combination on CLP-induced sepsis mice

[0040] 1 Experimental materials

[0041] 1.1 Medicinal materials and reagents

[0042] Medicinal materials and reagents Manufacturers Coptis (Batch No.: 20210101) Shaanxi Han Medicine Saint Grass Hall Pharmaceutical Co., Ltd. Gardenia (Batch No.: 22050805) An'guo City Guangjitang Pharmaceutical Co., Ltd. 0.9% Sodium chloride injection Chenxin Pharmaceutical Co., Ltd. 4% Tissue fixative Xianhete Biotechnology Co., Ltd. Isoflurane Hebei Jindafu Pharmaceutical Co., Ltd. Anhydrous ethanol National Pharmaceutical Group Chemical Reagent Co., Ltd. Xylene National Pharmaceutical Group Chemical Reagent Co., Ltd. Hematoxylin staining solution Wuhan Sevier Biological Technology Co., Ltd. Eosin staining solution Hefei Bomei Biological Technology Co., Ltd. Hydrochloric acid Chengdu Kolon Chemical Co., Ltd. Neutral gum Biosharp Biological Company Interleukin 6 (IL-6, Batch No. 202306) Jiangsu Enzyme-free Industry Co., Ltd. Tumor necrosis factor (TNF-α, Batch No. 202306) Jiangsu Enzyme-free Industry Co., Ltd. C-reactive protein (CRP, Batch No. 202306) Jiangsu Enzyme-free Industry Co., Ltd. Procalcitonin (PCT, Batch No. 202306) Jiangsu Enzyme-free Industry Co., Ltd. Endothelin (ET-1, Batch No. 202306) Jiangsu Enzyme-free Industry Co., Ltd. Glutamic-oxaloacetic transaminase (AST, Batch No. 202306) Jiangsu Enzyme-free Industry Co., Ltd. Glutamic-pyruvic transaminase (ALT, Batch No. 202306) Jiangsu Enzyme-free Industry Co., Ltd.

[0043] 1.2 Instruments and equipment

[0044] Instruments and equipment Manufacturers RE-52AA rotary evaporator Shanghai Yalong Biochemical Instrument Factory Mettler MS-105DU electronic analytical balance Switzerland Mettler-Toledo Company Multifunctional enzyme reader BioTek Company of the United States Constant temperature oscillator Shanghai Zhi Cheng Analytical Instrument Manufacturing Co., Ltd. Microfuge 20R high-speed refrigerated centrifuge Beckman Coulter Company Milli-Q IQ7000 ultrapure water system Millipore Company of the United States DHG-9140A air drying oven Shanghai Huite Instrument Manufacturing Co., Ltd. KE0003087 / KA0056573 pipettor Beijing Dalong Technology Co., Ltd. Rotary microtome Germany Leica Instrument Co., Ltd. Automatic dehydration machine Wuhan Junjie Electronics Co., Ltd. Tissue embedding machine Changzhou Qiaoxian Zhongwei Electronic Instrument Factory Pathological tissue bleaching and drying instrument Changzhou Zhongwei Electronic Instrument Co., Ltd. Digital three-eye camera microscope Mcody Industrial Group Co., Ltd.

[0045] 1.3 Experimental animals

[0046] 7-8-week-old male C57BL / 6 mice weighing 20-25 g, SPF level, provided by the Animal Experimental Center of Shaanxi University of Chinese Medicine, license number: SCXK(Shan)2021-001, light and dark periods of 12 h, humidity of 40%-60%, free water and food during the period. All experimental procedures were approved by the Animal Ethics Committee of Shaanxi University of Chinese Medicine, and the ethics review number was SUCMDL20230706001. The animals were adaptively fed for 7 days, and all animals were mixed in larger breeding cages, and half of the animals were randomly exchanged between the breeding cages every day.

[0047] 2 Experimental methods

[0048] 2.1 Preparation of liquid medicine

[0049] Take 200 g of Huanglian and Rhizoma Japonicae decoction pieces respectively, soak in 8 times the amount of water for 2 h, reflux extract 2 times, 1.5 h each time, filter through five layers of gauze, combine the two filtrates, concentrate and freeze-dry; Huanglian extract and Rhizoma Japonicae extract are obtained respectively.

[0050] When grouping, Huanglian extract and Rhizoma Japonicae extract are respectively combined according to the mass ratio of 5:9 and 9:9 to obtain.

[0051] 2.2 Experimental animal grouping

[0052] After one week of adaptive feeding, the mice were randomly divided into 8 groups: Sham group (sham operation group); CLP group (model group); Huanglian extract (LH; 0.65 g / kg); Huanglian extract (HH; 1.17 g / kg); Zhizi extract (ZZ; 1.17 g / kg); mass ratio 5:9 Huanglian-Zhizi group (LD; 1.17 g / kg); mass ratio 9:9 Huanglian-Zhizi group (HD; 1.17 g / kg); Xuebijing group (PC; 10 mL / kg). The mice in the Xuebijing group were given intraperitoneal injection, and the mice in the other groups were given gavage, for 7 consecutive days. The mice in the sham operation group and the model group were given pure water of the corresponding volume. Two hours after the last administration, the cecal ligation puncture (CLP) operation was performed to model. The modeling method was referred to Michael Bauer, Herwig Gerlach, Tobias Vogelmann, Franziska Preissing, Julia Stiefel, Daniel Adam. Mortality in sepsis and septic shock in Europe, North America and Australia between 2009 and 2019 - results from a systematic review and meta-analysis. Critical Care, 2020, 24(1): 239. article. 12 hours before the operation, the mice were fasted but not deprived of water, and they were free to eat and drink after the operation. The mice were placed in an induction box, the gas flow was adjusted to 1 L / min, and the isoflurane concentration was adjusted to 3%. After the mice lost the righting reflex, they were taken out of the induction box and placed on a fixation plate with their heads away from the operator. Anesthesia was maintained by connecting a mask to the animal's back and adjusting the isoflurane concentration to 1.2%. An electric trimmer was used to shave the lower half of the abdomen, and the area was disinfected with iodophor. A 1.5-2 cm incision was made in the middle of the abdomen. The cecum was further exposed, and the feces stored in the cecum were gently pushed to the top of the cecum. The cecum was ligated at a distance of one quarter from the bottom of the cecum (just below the ileocecal junction) with a sterile 4-0 thread, and a mild sepsis model was constructed. Avoid intestinal obstruction, and use a needle to puncture one hole. The cecum was gently compressed, and a small amount of cecal contents was squeezed out, and then the cecum was returned to the abdominal cavity. The abdominal incision was closed with 6-0 layered suture. After the operation, all mice were subcutaneously injected with 1 mL of preheated sodium chloride solution to prevent dehydration, and the mice were placed under a heating lamp to increase their temperature. The mice in the sham operation group also underwent laparotomy and intestinal manipulation (the cecum was neither ligated nor perforated). All surgical procedures were performed in accordance with the procedures specified by the Animal Ethics Committee. Enucleation was performed 24 hours after modeling to obtain serum from each group, and the mice were immediately executed by cervical dislocation after blood collection. The mice were dissected, and the liver, lung tissue, and cecal contents were taken for subsequent testing.

[0053] 2.3 Detection of biochemical indicators

[0054] The levels of IL-6, TNF-a, CRP, PCT, ET-1, AST and ALT in serum were determined according to the requirements of the enzyme-linked immunosorbent assay (ELISA) kit instructions.

[0055] 2.4 Liver and lung histopathological examination

[0056] The degree of liver and lung cell damage was observed by H&E staining. The liver and lung tissues of mice in each group were fixed in tissue fixative, routinely paraffin-embedded, sectioned, and observed under a light microscope after H&E staining to observe the pathological state.

[0057] 2.5 Data analysis

[0058] GraphPad Prism 8.3.0 statistical software was used for data processing, and all experimental data were expressed as "mean ± standard deviation". The data between two groups were compared by t-test, and the comparison between groups was performed by one-way ANOVA. p<0.05, indicating that the difference was statistically significant.

[0059] 3 Experimental results

[0060] 3.1 Effect of Coptis-Chrysanthemum Combination on the survival rate of sepsis mice

[0061] During the drug intervention process, the mice in each group had no obvious abnormal behavior, normal diet, formed feces, and were in good condition. Except for the sham operation group, the mice in the other groups showed symptoms such as body curling, reduced food intake, poor stress tolerance, reduced activity, increased eye secretions, most mice huddling for warmth, and sticky defecation after CLP. The 24h survival rate of mice was observed (statistical analysis was performed every 6h), and the survival status of mice was as shown in Figure 1 The 24h survival rate of mice in each group was: CLP group (60%), LH group (69.23%), HH group (86.67%), ZZ group (78.57%), LD group (100%), HD group (84.62%), and PC group (93.33%). The results showed that compared with the sham operation group, the survival rate of mice in the CLP group was significantly reduced (p<0.01), and the 24h survival rate of mice in the LD and PC groups was significantly higher than that in the model group (p<0.05).

[0062] 3.2 Effect of Coptis-Chrysanthemum Combination on the levels of IL-6, TNF-a, ET-1, PCT, CRP, AST and ALT in the serum of sepsis mice

[0063] The results of the following seven indicators show that compared with the Sham group, the levels of IL-6, TNF-a, ET-1, PCT, CRP, AST and ALT in the Model group were significantly increased (p<0.001; p<0.05). Compared with the model group, the indicators of mice in the administration group were basically improved, among which the levels of IL-6, TNF-a and ALT in the LH group were significantly reduced (p<0.001; p<0.05), the levels of ET-1, PCT, CRP and AST were reduced to a certain extent but had no significant difference; the levels of IL-6, TNF-a, ET-1, PCT and CRP in the HH group were significantly reduced (p<0.001; p<0.01), the levels of AST and ALT were reduced but had no significant difference; the levels of IL-6, TNF-a, ET-1, PCT and CRP in the ZZ group were significantly reduced (p<0.001; p<0.01; p<0.05), the levels of AST and ALT were reduced but had no significant difference; the levels of IL-6, TNF-a, ET-1, CRP, PCT, AST and ALT in the LD group were significantly reduced (p<0.001; p<0.01); the levels of IL-6, TNF-a, ET-1, CRP, PCT, AST and ALT in the HD group were significantly reduced (p<0.001; p<0.01; p<0.05); the levels of IL-6, TNF-a, ET-1, CRP, PCT and ALT in the PC group were significantly reduced (p<0.001; p<0.01; p<0.05), the level of AST was reduced to a certain extent but had no significant difference, and the specific results are shown in Table 2. Figure 2 In summary, the results show that Coptis and Gardenia can alleviate the inflammatory response of sepsis to a certain extent, and the effect is more significant after being matched, among which the LD group has the best effect.

[0064] 3.3 Effect of Coptis-Gardenia combination on the morphology of liver and lung tissues of sepsis mice

[0065] H&E staining section of liver tissue: The liver tissue structure of mice in the Sham group was normal, and the liver cells were arranged in cord-like, with clear boundaries. Compared with the Sham group, the liver tissue of mice in the CLP group had relatively severe lesions, and obvious hepatocyte steatosis was observed. Compared with the CLP group, the liver tissue of mice in the LH group, HH group, ZZ group, HD group and PC group showed slight hepatocyte steatosis, in addition, the LD group showed a very small amount of hepatocyte steatosis, and the results are shown in Figure 3. Figure 3 .

[0066] Lung tissue H&E staining section: The surface of the lung tissue of the sham group mice was covered with serosa, and there was no edema, inflammatory infiltration or fibrous connective tissue proliferation. The structure of the bronchus at all levels was complete and clear, and the bronchial epithelial cells were normal. Compared with the sham group, the lung of the CLP group mice was obviously degenerated, a small amount of alveolar epithelial cells were degenerated and necrotic, the necrotic cells were disintegrated and fragmented, the alveolar cavity was at different degrees of atrophy, and the interstitial inflammatory cell infiltration was mainly in the form of rod-shaped segmented neutrophils. Compared with the CLP group, the lung tissue of the rest of the groups could alleviate the damage to the lung tissue to a certain extent. The lung tissue of the LH group showed slight alveolar cavity atrophy and inflammatory cell infiltration, the lung tissue of the HH group showed only mild alveolar cavity atrophy, the lung tissue of the ZZ group, the HD group and the PC group showed slight inflammatory cell infiltration, and in addition, the lung tissue of the LD group showed no obvious pathological changes. In summary, the combination of Coptis and Gardenia can significantly improve the damage to the liver and lung tissue caused by sepsis, and the LD group is the most effective. The results are shown in Figure 4 .

[0067] One of the core pathogenesis of sepsis is uncontrolled inflammatory response, which is characterized by excessive activation of inflammatory cells and release of large amounts of inflammatory cytokines. In this study, CLP was used to construct a sepsis mouse model. Based on this model, a series of key biomarkers were selected to evaluate the inflammatory response, infection and liver cell damage. IL-6 as a biomarker of survival and disease status in CLP model, its high level is closely related to the high risk of severe sepsis and mortality, and is an early sensitive early warning indicator. TNF-α plays a direct role in the development of septic shock by inducing apoptosis and participating in immune suppression, etc. PCT can be used as a serum marker for bacterial infection, which has important significance for the detection of sepsis, and is an important factor for judging the existence and severity of infection in clinic. CRP is one of the most commonly used biomarkers for early diagnosis of sepsis in clinic, and is also a widely used inflammatory marker. PCT and CRP are the most widely used biomarkers for sepsis research, followed by IL-6 and endothelin. Studies have shown that the combined use of PCT, CRP and IL-6 can improve the diagnostic value of single detection. In addition, AST and ALT are the most commonly used liver function detection indicators. Therefore, this experiment used ELISA to detect the levels of IL-6, TNF-α, ET-1, PCT, CRP, AST and ALT in serum to evaluate the degree of inflammation, infection and liver cell damage in sepsis mice induced by CLP. The results showed that the levels of IL-6, TNF-α, ET-1, PCT, CRP, AST and ALT in serum of sepsis mice were significantly increased, while the levels of IL-6, TNF-α, ET-1, PCT, CRP, AST and ALT in serum of Huanglian and Zhizi administration groups were decreased, indicating that Huanglian and Zhizi administration groups had a certain protective effect on sepsis mice, and the effect of Huanglian and Zhizi combination was better, and the effect of LD group was the most significant.

[0068] In addition, sepsis can cause organ dysfunction, to directly assess the protective effect of Huanglian Zhizi couplet, this study on the liver and lung tissue of each group of mice were stained with H&E analysis. The results show that, compared with the Sham group, CLP group of mice liver and lung tissue showed typical pathological changes: liver tissue can be seen extensive hepatocyte steatosis, while lung tissue alveolar epithelial cell degeneration and necrosis, cell disintegration, fragmentation, with varying degrees of alveolar space collapse and interstitial inflammatory cell infiltration, mainly with rod-shaped lobed neutrophils. Indicate that mild sepsis model of success. Huanglian, Zhizi single drug and compatibility of mice, liver and lung tissue damage were significantly eased. Among them, Huanglian Zhizi compatibility group showed a more optimal protective effect, especially in the LD group, the degree of histopathological damage was significantly reduced. The above results show that Huanglian Zhizi couplet can effectively prevent sepsis in mice liver and lung tissue pathological damage, reduce the inflammatory response and infection, for its clinical application in sepsis treatment provides an important histological basis.

[0069] Example 2 Huanglian Zhizi Compatibility of CLP-induced sepsis in mice comparative metabonomics study 1 experimental instruments and reagents

[0070]

[0071]

[0072] 2 Method

[0073] 2.1 Serum sample processing method

[0074] Take the serum samples of each group in Example 1, after taking out from the-80℃ refrigerator, thawing at 4℃, take 50μL serum and add 300μL pre-cooled methanol, vortex 1min, 4℃, 12000rpm, centrifugal 15min, remove 200μL supernatant, vacuum centrifugal concentration to dryness, add 200μL 10% cold acetonitrile (acetonitrile: water = 1:9, v / v) for re-dissolution, vortex and centrifugal again, take the supernatant into analysis. From each serum sample, 20μL mixed into quality control samples (QC), and processed as above. Every 10 samples inserted a QC, to test the stability of the system.

[0075] 2.2 Metabonomics analysis

[0076] 2.2.1 Detection conditions

[0077] Chromatographic conditions: ACQUITY UPLC HSS C18 column (2.1 mm x 100 mm, 1.8 μm), mobile phase: 0.1% formic acid aqueous solution (A) - acetonitrile (B), flow rate 0.30 mL / min, column temperature 35 °C, injection volume 3 μL. Elution conditions: 0-3.0 min 95-85% A, 3-6 min 85-78% A, 6-13 min 78-5% A, 13-16 min 5-0% A, 16-17 min 0% A, 17-18 min 0-95% A, 18-20 min 95% A.

[0078] Mass spectrometry conditions: in positive ion mode, capillary voltage 3.0 KV, cone hole voltage 55 V, desolvation gas temperature 350 °C, desolvation gas flow 800 L / h, ion source temperature 100 °C, cone hole gas flow 50 L / h. The scan range was m / z 50-1200 Da.

[0079] 2.2.1 Data preprocessing

[0080] Progenesis QI: The raw data of Waters instrument (.raw) was imported into Progenesis QI software for peak alignment, peak extraction, peak identification and normalization processing, and EZinfo 3.0 software was used for data analysis.

[0081] MS-DIAL: The raw data of Waters instrument (.raw) was imported into MSConvertGUI software, and converted into (.mzML) format files. Then, these files were imported into MS-DIAL ver 4.24, and subsequent operations were performed through data collection, peak extraction, identification, normalization and other steps. Combined with the results of the two software, the differential metabolites with VIP>1 and p<0.05 were selected for subsequent analysis.

[0082] 2.3 Identification and pathway analysis of differential metabolites

[0083] Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used for data analysis, and metabolites with VIP > 1 and p < 0.05 were selected as potential biomarkers. The accurate m / z and retention time were obtained by UPLC-Q-TOF / MS, and the corresponding database (Progenesis QI, MSDIAL, HMDB) was searched for mass spectrum information matching. The potential biomarkers were preliminarily identified according to the actual fragment ions and theoretical fragment ions, and the identified markers were introduced into MetaboAnalyst (http: / / www.metaboanalyst.ca) for enrichment and analysis of metabolic pathways. Graphpad Prism 8.3.0 software was used for statistical analysis. p < 0.05, the difference was statistically significant.

[0084] 3. Results of the experiment

[0085] 3.1 UPLC-Q-TOF / MS method validation and serum metabolic profile

[0086] Figure 5 Figures 4A and 4B are BPI diagrams in positive ion mode of representative serum samples collected from the Sham group and the CLP group, respectively. Figure 6 Figures 6A and 6B are the results of PCA and OPLS-DA score plots showing a clear separation between the two groups, indicating that the metabolic characteristics have changed significantly. The parameters R 2 Y = 0.985 and Q 2 = 0.969, R 2 Y is used to evaluate the fitness of the model, and Q 2 is used to evaluate the predictive ability of the model, and the results show that the model has good predictive and explanatory ability, and the model is reliable. In addition, Figure 6 Figures 6C and 6D are the results of the orthogonal projection of the PCA and PLS-DA score plots, showing that the metabolic characteristics of the five groups are clearly distinguished.

[0087] 3.2 Screening of differential metabolites

[0088] VIP>1 and p<0.05 as screening conditions, the greater the difference between these metabolites, the more likely to be potential differential metabolites of sepsis. A total of 59 differential metabolites were screened between Sham vs CLP group, on this basis, 42 common differential metabolites were screened between Sham vs CLP and CLP vs LD group, among which Huanglian-Zhizi (5:9) could reverse 32 differential metabolites; 40 common differential metabolites were screened between Sham vs CLP and CLP vs HD group, among which Huanglian-Zhizi (1:1) could reverse 24 differential metabolites; 26 common differential metabolites were screened between Sham vs CLP and CLP vs PC group, among which Xuebijing injection could reverse 11 differential metabolites; In addition, it was found that there were 32 common differential metabolites between Sham vs CLP, CLP vs LD and CLP vs HD group, among which Huanglian-Zhizi of different proportions could reverse 17 differential metabolites (Table 1). Through cluster analysis, the relationship between 17 metabolites between the four groups was visualized based on heat map. Figure 7 These metabolite components were increased or decreased in the CLP group compared with the Sham group, and after the administration of Huanglian-Zhizi, the level of metabolites was significantly reversed, among which the LD group (Huanglian-Zhizi 5:9) had more obvious regulation effect.

[0089] Table 1 Common differential metabolites between serum samples of Sham group, CLP group, LD group and HD group

[0090]

[0091]

[0092] 3.3 Metabolic pathway analysis

[0093] The reversed differential metabolites of each comparison group were uploaded to the MetaboAnalyst 6.0 database for metabolic pathway enrichment analysis. The results showed that: the reversed metabolites of Sham vs CLP and CLP vs LD were enriched in 26 metabolic pathways, of which 17 pathways had an Impact > 0, mainly involving amino acid metabolism, lipid metabolism, purine metabolism, glycan biosynthesis and metabolism, metabolism of cofactors and vitamins, and metabolism of other amino acids; the reversed metabolites of Sham vs CLP and CLP vs HD were enriched in 20 metabolic pathways, of which 11 pathways had an Impact > 0, mainly related to amino acid metabolism, lipid metabolism, metabolism of cofactors and vitamins, glycan biosynthesis and metabolism, and metabolism of other amino acids; the reversed metabolites of Sham vs CLP and CLP vs PC were enriched in 9 metabolic pathways, of which 6 pathways had an Impact > 0, mainly related to lipid metabolism, metabolism of other amino acids, and glycan biosynthesis and metabolism; the reversed differential metabolites of Sham vs CLP, CLP vs LD, and CLP vs HD groups were enriched in 16 metabolic pathways (Table 2), of which 8 pathways had an Impact > 0. These metabolic pathways were mainly related to amino acid metabolism and lipid metabolism. Metabolic pathway analysis further confirmed that amino acid metabolism and lipid metabolism were the key pathways of Huanglian-Jizhi drug pair against sepsis serum metabolism, and the specific pathways are shown in Figure 8 .

[0094] Table 2 Differential metabolic pathways related to sepsis resistance of Huanglian-Jizhi drug pair

[0095]

[0096]

[0097] In this study, we analyzed the small molecule metabolites in the serum of sepsis mice by metabolomics technology. The results showed that the contents of differential metabolites in the serum of sepsis mice were significantly changed compared with the sham operation group. After the intervention of Huanglian-Jizhi drug pair, the phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, alanine, aspartate and glutamate metabolism, arginine and proline metabolism, glycerophospholipid metabolism, primary bile acid biosynthesis, steroid hormone biosynthesis and steroid biosynthesis metabolic pathways were significantly regulated. These metabolic pathways play a key role in the serum metabolic regulation of Huanglian-Jizhi drug pair against sepsis, revealing the potential targets and mechanisms of the drug in the intervention of sepsis metabolic disorder.

[0098] Sepsis is a major clinical problem, its pathological process involves complex mechanisms such as uncontrolled inflammatory response, immune dysfunction and metabolic disorders. Among them, amino acid metabolism plays a central role in the regulation of adaptive immunity and innate immunity. Not only does it regulate immune cell activation and antibody production, but the changes in its metabolic state are also closely related to disease outcome. Sepsis-induced insulin resistance, glucose and lipid metabolism disorders, and liver function damage often lead to imbalances in amino acid metabolism. Studies have shown that bacterial infection (such as sepsis) can significantly disrupt amino acid metabolism, especially the imbalance of aromatic amino acid pathway. For example, L-phenylalanine may be accelerated to decompose or transform into tyrosine, phenethylamine and other metabolites under the condition of sepsis, resulting in a decrease in serum levels. Both Huanglian and Zhizi have anti-inflammatory effects, and after being combined, they may alleviate the metabolic disorder caused by sepsis by inhibiting the release of inflammatory mediators, and then restore the normal metabolism of L-phenylalanine. As the most abundant amino acid in the human body, glutamine showed characteristic changes in this study: the content of glutamine in the serum of the sham operation group was low, and its content in the serum of the model group increased after modeling. After the intervention of Huanglian-Zhizi drug pair, glutamine showed a tendency of adjustment, which is consistent with previous studies. In addition, muscle protein degradation caused by sepsis will affect the supply of branched-chain amino acids. This study found that the level of L-valine in mice treated with Huanglian-Zhizi (5:9) was lower. This suggests that the drug pair may play a protective role in sepsis by regulating amino acid metabolism.

[0099] Disorders of lipid metabolism are also important pathological features of sepsis, often accompanied by dyslipidemia and liver damage. This study found that Huanglian-Zhizi drug pair intervention can effectively reverse the downward trend of serum LysoPC levels in sepsis mice. Endogenous LysoPC not only enhances the ability to clear the abdominal cavity of bacteria and blocks neutrophil inactivation, but also significantly reduces the mortality of sepsis mice by improving the bactericidal activity of neutrophils. At the level of fatty acid metabolism, palmitic acid, as a marker of liver damage and a key factor in fatty acid synthesis, can help improve liver damage. At the same time, oleic acid can reduce organ dysfunction and mortality by promoting fatty acid oxidation. Compared with the CLP group, the levels of oleic acid and palmitic acid in the Huanglian-Zhizi (5:9) combination group were higher than those in the model group, which helped to alleviate the hypermetabolic state of sepsis mice. The Huanglian-Zhizi (1:1) combination group showed the opposite trend. This result further confirmed that Huanglian-Zhizi (5:9) combination prophylactic administration has a significant protective effect on liver damage in sepsis mice. Huanglian-Zhizi drug pair intervention increased the expression level of docosahexaenoic acid in the serum of sepsis mice, suggesting its potential value in inflammation regulation.

[0100] In summary, this study found that different ratios of Huanglian-Zhizi drug pair intervention have different regulatory effects on metabolites, and can effectively prevent the disorder of metabolic pathways such as amino acid metabolism, lipid metabolism, energy metabolism, and carbohydrate metabolism caused by sepsis, and to a certain extent, play an anti-sepsis role.

[0101] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. Use of a Huanglian-Zhizi pair in the preparation of a medicine for resisting sepsis.

2. Use according to claim 1, characterized in that, The mass ratio of Huanglian and Zhizi is 5:5-5:

9.

3. Use according to claim 1, characterized in that, The extraction method of Huanglian and Zhizi is as follows: Huanglian and Zhizi decoction pieces are taken in a mass ratio of 5:5-5:9, soaked in 8-12 times the amount of water for 0.5-1 h, reflux extracted 2-3 times, each time for 0.5-1.5 h, filtered, the filtrates are combined, concentrated, and then freeze-dried to obtain the extract of the Huanglian-Zhizi pair.

4. Use according to claim 1, characterized in that, The extraction method of Huanglian and Zhizi is as follows: Huanglian and Zhizi decoction pieces are taken in a mass ratio of 5:5-5:9, soaked in 8-12 times the amount of water for 0.5-1 h, reflux extracted 2-3 times, each time for 0.5-1.5 h, filtered, the filtrates are combined, concentrated, and then freeze-dried to obtain the extract of the Huanglian-Zhizi pair.

5. The use according to claim 1, wherein the metabolomics research method of the Huanglian-Zhizi pair for resisting sepsis comprises the following steps: (1) Animal grouping and administration After adaptive feeding for one week, the mice are randomly divided into 8 groups, namely a sham operation group, a model group, a Huanglian LH group, a Huanglian HH group, a Zhizi ZZ group, a Huanglian-Zhizi mass ratio of 5:9 group, a Huanglian-Zhizi mass ratio of 9:9 group, a Xuebijing group, and a Xuebijing group of mice is intraperitoneally injected, and the rest of the mice is orally administered, continuously for 7 days; the sham operation group and the model group of mice are given pure water of a corresponding volume; 2 h after the last administration, a cecal ligation and puncture operation is performed to model; 24 h after modeling, the mice are enucleated to take blood, and the serum samples are stored in a -80℃ refrigerator; immediately after blood collection, the mice are executed by cervical dislocation, and the mice are dissected to take liver, lung tissue and cecal contents for subsequent detection; (2) Serum sample processing method After being taken out of the -80℃ refrigerator, the serum samples are thawed at 4℃, and serum is added to pre-cooled methanol, vortexed, centrifuged, and the supernatant is transferred, vacuum centrifuged to dryness, and then re-dissolved in a cold acetonitrile water solution, vortexed and centrifuged again to obtain the supernatant sample for analysis; (3) Metabolomics analysis The serum samples of each group in step (2) are subjected to UPLC-Q-TOF / M metabolomics analysis; (4) Data preprocessing Progenesis QI: The raw data of the Waters instrument is imported into the Progenesis QI software for peak alignment, peak extraction, peak identification and normalization processing, and the EZinfo 3.0 software is used for data analysis; MS-DIAL: The raw data of the Waters instrument is imported into the MSConvertGUI software, and converted into.mzML format files; then, these files are imported into MS-DIALver 4.24, and subsequent operations are performed through data collection, peak extraction, identification, normalization steps; combined with the results of the two software, the differential metabolites with VIP>1 and p<0.05 are selected for subsequent analysis; (5) Identification and pathway analysis of differential metabolites The data were analyzed by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA), and the metabolites with VIP>1 and p<0.05 were selected as potential biomarkers; the accurate m / z and retention time were obtained by UPLC-Q-TOF / MS, and the corresponding database Progenesis QI, MSDIAL, HMDB was searched for mass spectrum information matching; the potential biomarkers were preliminarily identified according to the actual fragment ions and theoretical fragment ions, and the identified markers were imported into MetaboAnalyst (http: / / www.metaboanalyst.ca) for enrichment and analysis of metabolic pathways; statistical analysis was performed using Graphpad Prism 8.3.0 software.

6. Use according to claim 5, characterized in that, Step (1) animal grouping and administration After one week of adaptive feeding, the mice were randomly divided into 8 groups, namely, sham operation group; model group; Huanglian LH group, dose 0.65 g / kg; Huanglian HH group, dose 1.17 g / kg; Zhizi ZZ group, dose 1.17 g / kg; Huanglian-Zhizi mass ratio 5:9 group, dose 1.82 g / kg; Huanglian-Zhizi mass ratio 9:9 group, dose 2.34 g / kg; Xuebijing group, 10 mL / kg; Xuebijing group mice were given intraperitoneal injection, and the rest of the mice were given oral administration, for 7 consecutive days; the sham operation group and the model group mice were given pure water of corresponding volume; 2 hours after the last administration, the cecal ligation and puncture operation modeling was performed; 24 hours after modeling, the mice were enucleated and blood was taken, and the serum samples were stored at -80°C in the refrigerator; immediately after blood collection, the mice were executed by cervical dislocation, dissected, and the liver, lung tissue and cecal contents were taken for subsequent detection.

7. Use according to claim 5, characterized in that, Step (2) serum sample processing method After taking out the serum sample from the -80°C refrigerator, it was thawed at 4°C, 50 μL of serum was added to 300 μL of pre-cooled methanol, vortexed for 1 min, centrifuged at 4°C, 12000 rpm for 15 min, 200 μL of supernatant was removed, vacuum centrifuged to dryness, and then 200 μL of 10% cold acetonitrile aqueous solution was added for reconstitution, vortexed and centrifuged again to obtain the supernatant sample for analysis.

8. Use according to claim 5, characterized in that, Step (3) Chromatographic conditions for metabolomics analysis: ACQUITY UPLC HSS C18 column, specifications 2.1 mm x 100 mm, 1.8 μm, mobile phase 0.1% formic acid aqueous solution as A phase-acetonitrile as B phase, flow rate 0.30 mL / min, column temperature 35°C, injection volume 3 μL; elution conditions: 0-3.0 min 95%-85% A, 3-6 min 85%-78% A, 6-13 min 78%-5% A, 13-16 min 5%-0% A, 16-17 min 0% A, 17-18 min 0%-95% A, 18-20 min 95% A; Mass spectrometry conditions: In positive ion mode, the capillary voltage was 3.0 KV, the cone hole voltage was 55 V, the desolvation gas temperature was 350℃, the desolvation gas flow was 800 L / h, the ion source temperature was 100℃, and the cone hole gas flow was 50 L / h. The scanning range was m / z 50-1200 Da.

9. Use according to claim 5, characterized in that, The differential metabolites were Glutamine, Octadecanedioic acid, LysoPC(18:1(9Z) / 0:0), Icosanoic acid, Creatine, Cholesterol, Docosanoic acid, Glycocholic acid, Docosahexaenoic acid, Tetracosanoic acid, 4-Coumaric acid, L-Phenylalanine, Caffeic acid, L-Methioninesulfone, 9-Octadecenal, L-Carnitine, 5-Methoxytryptophol.

10. Use according to claim 5, characterized in that, Huanglian-Zhizi pair plays a role in resisting sepsis through phenylalanine metabolism, arginine and proline metabolism, and glycerophospholipid metabolism pathways.

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