A traditional Chinese medicine composition for preventing and treating sepsis gastrointestinal dysfunction and a preparation method thereof

Abstract

The application discloses a traditional Chinese medicine composition for preventing and treating sepsis gastrointestinal dysfunction and a preparation method thereof. Raw materials are composed of the following components in parts by weight: 7-10 parts of red ginseng, 7-10 parts of prepared aconite root tablets, 5-7 parts of Schisandra chinensis, 7-10 parts of raw rhubarb, 11-13 parts of coix seed and 11-13 parts of Herba Patriniae. Experimental results show that the application has good clinical curative effect and pharmacological action on treating sepsis gastrointestinal dysfunction.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition for preventing and treating sepsis gastrointestinal dysfunction and a preparation method. BACKGROUND

[0002] Sepsis is a fatal organ dysfunction or failure caused by the disorder of host response to infection. Epidemiological investigations show that there are still about 31.5 million sepsis patients worldwide every year, and about 5.3 million people die of sepsis, which is still on the rise. The pathogenesis of sepsis is complex, and the occurrence and development are closely related to the pathophysiological changes of multiple systems and multiple important organs of the human body. Research has found that the gastrointestinal tract is not only one of the most vulnerable target organs in the pathogenesis of sepsis, but also a pivotal organ in the occurrence and development of multiple organ dysfunction syndrome (MODS). Uncontrolled inflammatory response, circulatory disorder and immunosuppression during sepsis can all lead to gastrointestinal dysfunction, and the damage of mucosal barrier caused by gastrointestinal dysfunction can further aggravate the injury of various organs, causing a "second hit" and forming a vicious cycle. A large amount of evidence shows that gastrointestinal dysfunction is the most vulnerable to damage in the progression of sepsis, and is a major independent risk factor for the prognosis of sepsis patients.

[0003] The concept of "preventing disease before disease occurs" and "treating disease by seeking the root" of traditional Chinese medicine has unique advantages and characteristics. There is no clear name for gastrointestinal dysfunction in traditional Chinese medicine, and according to the symptoms such as abdominal pain, abdominal distension, poor appetite, constipation and vomiting, it is classified into "distention and fullness", "abdominal pain", "constipation" and "vomiting". Spleen and stomach are the acquired basis of human body and the source of qi and blood, and are the key to resist the invasion of evil. If the spleen yang is deficient and the body's resistance to evil is decreased, the evil will enter the body, and the evil and the healthy will fight each other, and then the qi, blood, yin and yang will be in disorder, and finally the function of viscera will be declined. At present, there is no good traditional Chinese medicine compound composition for treating sepsis gastrointestinal dysfunction.

[0004] Therefore, the present application is proposed. SUMMARY

[0005] The present application aims to overcome the above technical deficiencies, and provides a traditional Chinese medicine composition for preventing and treating sepsis gastrointestinal dysfunction and a preparation method, which has good clinical efficacy and pharmacological effect on sepsis gastrointestinal dysfunction.

[0006] To achieve the above technical purpose, the present application adopts the following technical scheme:

[0007] According to one aspect of the present application, a traditional Chinese medicine composition for preventing and treating sepsis gastrointestinal dysfunction is provided, and the raw materials are composed of the following components by weight parts: 7-10 parts of red ginseng, 7-10 parts of prepared cortex phellodendri, 5-7 parts of Schisandra chinensis, 7-10 parts of raw rhubarb, 11-13 parts of coix seed and 11-13 parts of Herba Patriniae.

[0008] Further, the weight parts of each component are 9 parts of red ginseng, 9 parts of prepared aconite, 6 parts of Schisandra chinensis, 9 parts of raw rhubarb, 12 parts of coix seed and 12 parts of patrinia.

[0009] Further, the weight parts of each component are 7 parts of red ginseng, 10 parts of prepared aconite, 5 parts of Schisandra chinensis, 10 parts of raw rhubarb, 11 parts of coix seed and 13 parts of patrinia.

[0010] Further, the weight parts of each component are 10 parts of red ginseng, 7 parts of prepared aconite, 7 parts of Schisandra chinensis, 7 parts of raw rhubarb, 13 parts of coix seed and 11 parts of patrinia.

[0011] The application also provides a preparation method of the traditional Chinese medicine composition for preventing and treating sepsis gastrointestinal dysfunction, which comprises the following steps:

[0012] The six medicinal decoction pieces of prepared aconite, red ginseng, rhubarb, coix seed, Schisandra chinensis and patrinia are weighed according to the proportions in the traditional Chinese medicine composition of claim 1, washed and dried.

[0013] The above decoction pieces are washed and put into containers respectively for standby; the prepared aconite is first decocted for 0.5 h, then the four decoction pieces of rhubarb, coix seed, Schisandra chinensis and patrinia are added and decocted twice, 8 times water is added for the first time and decocted for 1.5 h, and the precipitate is filtered; 6 times water is added to the residue for the second time and decocted for 1 h, and the precipitate is filtered again; the decoction liquids of the two times are combined, the liquid is concentrated, cooled at room temperature, and the first decoction liquid is obtained for standby;

[0014] The red ginseng is decocted twice, 8 times water is added for the first time and decocted for 1.5 h, and the precipitate is filtered; 6 times water is added to the residue for the second time and decocted for 1 h, and the precipitate is filtered again; the decoction liquids of the two times are combined, the liquid is concentrated, cooled at room temperature, and the second decoction liquid is obtained for standby; the first decoction liquid and the second decoction liquid are combined and concentrated.

[0015] The traditional Chinese medicine composition provided by the application is collectively referred to as Weichangfuyuan recipe in the following for convenience, which is composed of prepared aconite, red ginseng, rhubarb, coix seed, Schisandra chinensis and patrinia. The two drugs of red ginseng and aconite are combined to form Shenshu decoction, which is used as a monarch drug and has the functions of restoring yang and astringing. Rhubarb is used as a minister drug to eliminate accumulated heat and toxic material in the intestines. Coix seed and patrinia are used as auxiliary drugs to increase the effects of clearing heat, resolving toxins, removing dampness, dispersing and resolving, and expelling pus. Aconite is used as a minister drug to invigorate yang. Schisandra chinensis is used as a minister drug to prevent the damage of rhubarb to yin fluid. The Weichangfuyuan recipe has reasonable composition, considers both symptoms and root causes, and adheres to the treatment principle of tonifying the spleen and stomach and combining tonification and purgation proposed by Professor Wei Beihai, a famous old doctor of traditional Chinese medicine in the capital.

[0016] Red ginseng (Panax ginseng C.A.Mey) is the dried root and rhizome of the cultivated product of Panax ginseng of Araliaceae, which is steamed. Compared with ginseng, red ginseng contains more types and contents of saponins, and has more abundant biological activities. Red ginseng has the effects of tonifying primordial qi, restoring pulse and preventing collapse, and is a main drug for tonifying primordial qi. Prepared aconite (Aconitum carmichaelii Debx) is the processed product of the suberect root of Aconitum carmichaelii of Ranunculaceae, which has the effects of restoring yang and saving from collapse, tonifying fire and assisting yang, and dispelling cold and relieving pain. Rhubarb (Rheum palmatum L) is the dried root and rhizome of Rheum palmatum L, Rheum webbianum or Rheum officinale, and has the effects of purgation, clearing heat and purging fire, cooling blood and detoxifying, removing blood stasis and unblocking channels, and removing dampness and relieving jaundice. Coix seed (Coix lacryma-jobi L.var.ma-yuen Stapf) is the dried mature seed of Coix lacryma-jobi L.var.ma-yuen Stapf of Poaceae, which has the effects of removing dampness and water, invigorating spleen and stopping diarrhea, removing obstruction, removing pus, detoxifying and resolving mass. Patrinia (Patrinia scabiosaefolia) is the whole herb with root of Patrinia scabiosaefolia or Patrinia villosa of Patrinia, which has the effects of clearing heat and detoxifying, removing pus and removing blood stasis. Schisandra (Schisandra chinensis.Baill) is the dried mature fruit of Schisandra chinensis.Baill of Magnoliaceae, which has the effects of astringing and tonifying, benefiting qi and producing saliva, and tonifying kidney and calming heart.

[0017] Modern pharmacological researches prove that the main chemical components of red ginseng are saponins, saccharides and volatile oils, and the pharmacological effects of red ginseng mainly include anti-aging, enhancing immunity and anti-oxidation. The main component of prepared aconite is aconite alkaloids, and aconitum base can participate in the immune response of the body by inhibiting the activation of NF-κB. The main components of rhubarb are anthraquinone derivatives and glycosides. Anthraquinone derivatives can reduce the positive rate of endotoxemia and the concentration of plasma endotoxin, inhibit the excessive activation of macrophages, and reduce the endotoxemia that may occur in acute infection. The production of interleukin-2 is inhibited by emodin, thereby regulating the immunity of the body. The effective component of coix seed, coix seed polysaccharide, has an inhibitory effect on early acute inflammation, and the anti-inflammatory effect is related to the inhibition of the exudation of inflammatory early tissue fluid. The effective component of patrinia, scabiosaefolia methanol, can play an anti-inflammatory effect by inhibiting the expression of related proteins in the NF-κB pathway such as transcription factor p65, TNF-α, IL-6 and cyclooxygenase COX-2. The main active component of schisandra is lignans. Lignans can increase the anti-apoptosis effect of tissues by promoting the activity of AKT.

[0018] Experiments prove that the application has good clinical curative effect on the treatment of sepsis gastrointestinal dysfunction.

[0019] Function and indication: invigorating the spleen, removing blood stasis. It is used for spleen deficiency, turbidity and toxin accumulation. Symptoms include abdominal distension, abdominal fullness, poor appetite, fatigue, cold, cold limbs, edema of lower limbs, dark complexion, even skin, tongue pale, thick white moss, and blood stasis on the tongue surface. Sepsis and gastrointestinal dysfunction (western medicine diagnosis) are also treated. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Figure 1 is a graph of HE staining of intestinal tissue of rats in each group (x100), wherein A is a blank group, B is a model group, C is a dexamethasone group, D is a low-dose gastrointestinal recovery prescription group, E is a medium-dose traditional Chinese medicine gastrointestinal recovery prescription group, and F is a high-dose traditional Chinese medicine gastrointestinal recovery prescription group.

[0021] Figure 2 Figure 2 is a graph of comparison of serum TNF-α, IL-6 and intestinal tissue IFN-γ, IL-1β levels of rats in each group.

[0022] Figure 3 Figure 3 is a graph of observation and comparison of mesenteric microcirculation of rats in each group, wherein A is a blank group, B is a model group, C is a dexamethasone group, D is a low-dose gastrointestinal recovery prescription group, E is a medium-dose traditional Chinese medicine gastrointestinal recovery prescription group, and F is a high-dose traditional Chinese medicine gastrointestinal recovery prescription group.

[0023] Figure 4 Figure 4 is a graph of comparison of PCNA expression of intestinal tissue of rats in each group (x100), wherein A is a blank group, B is a model group, C is a dexamethasone group, D is a low-dose gastrointestinal recovery prescription group, E is a medium-dose traditional Chinese medicine gastrointestinal recovery prescription group, and F is a high-dose traditional Chinese medicine gastrointestinal recovery prescription group.

[0024] Figure 5 Figure 5 is a graph of comparison of PCNA expression of intestinal tissue of rats in each group.

[0025] Figure 6 Figure 6 is a graph of comparison of apoptosis of intestinal tissue of rats in each group (x100), wherein A is a blank group, B is a model group, C is a dexamethasone group, D is a low-dose gastrointestinal recovery prescription group, E is a medium-dose traditional Chinese medicine gastrointestinal recovery prescription group, and F is a high-dose traditional Chinese medicine gastrointestinal recovery prescription group.

[0026] Figure 7 Figure 7 is a graph of comparison of apoptosis rate of intestinal tissue of rats in each group.

[0027] Figure 8A Figure 8 is a graph of comparison of expression of HSP90-α and Caspase-3 in intestinal tissue of rats in each group. A is a blank group, B is a model group, C is a dexamethasone group, D is a low-dose gastrointestinal recovery prescription group, E is a medium-dose traditional Chinese medicine gastrointestinal recovery prescription group, and F is a high-dose traditional Chinese medicine gastrointestinal recovery prescription group.

[0028] Figure 8BThe expression of HSP90-α and Caspase-3 in the intestinal tissue of each group of rats is compared in a columnar graph. DETAILED DESCRIPTION

[0029] In order to enable persons skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by persons skilled in the art without creative labor should fall within the protection scope of the present application.

[0030] Embodiment 1

[0031] The present application provides a traditional Chinese medicine composition for preventing and treating sepsis gastrointestinal dysfunction, which is prepared from raw materials of red ginseng, prepared aconite root, schisandra chinensis, raw rhubarb, coix seed and patrinia. The weight parts of each component are as follows: 9 parts of red ginseng, 9 parts of prepared aconite root, 6 parts of schisandra chinensis, 9 parts of raw rhubarb, 12 parts of coix seed and 12 parts of patrinia.

[0032] The preparation method is as follows: the prepared aconite root, red ginseng, rhubarb, coix seed, schisandra chinensis and patrinia are weighed according to the above-mentioned proportions, washed and dried.

[0033] The above-mentioned decoction pieces are washed and placed in containers for standby use. The prepared aconite root is first decocted for 0.5 h, and then the four decoction pieces of rhubarb, coix seed, schisandra chinensis and patrinia are added and decocted twice. The first time, 8 times the amount of water is added and decocted for 1.5 h, and the precipitate is filtered. The second time, 6 times the amount of water is added to the filtrate and decocted for 1 h, and the precipitate is filtered again. The decoction liquids of the two times are combined, the liquid is concentrated, and the second decoction liquid is obtained after cooling at room temperature.

[0034] Among them, the first decoction of aconite is mainly to reduce the toxicity of the medicine. The toxic component of aconite is aconitine compounds. The toxicity of these compounds will decrease after being heated with water. The toxicity of prepared aconite is much lower than that of raw aconite, but it still needs to be decocted for half an hour before being added to other traditional Chinese medicines for decoction.

[0035] The red ginseng is decocted twice with water. The first time, 8 times the amount of water is added and decocted for 1.5 h, and the precipitate is filtered. The second time, 6 times the amount of water is added to the filtrate and decocted for 1 h, and the precipitate is filtered again. The decoction liquids of the two times are combined, the liquid is concentrated, and the second decoction liquid is obtained after cooling at room temperature. The first decoction liquid and the second decoction liquid are combined and concentrated to obtain the traditional Chinese medicine composition.

[0036] Among them, the red ginseng medicine is relatively precious. Decoction alone and then mixed with other traditional Chinese medicines can effectively reduce the loss of components and maximize the efficacy of red ginseng.

[0037] Embodiment 2

[0038] The embodiment of the present application provides a traditional Chinese medicine composition for preventing and treating sepsis gastrointestinal dysfunction, raw materials of which are red ginseng, prepared aconite tuber, schisandra chinensis, raw rhubarb, coix seed and patrinia.

[0039] The preparation method is the same as that in Example 1.

[0040] Example 3

[0041] The embodiment of the present application provides a traditional Chinese medicine composition for preventing and treating sepsis gastrointestinal dysfunction, raw materials of which are red ginseng, prepared aconite tuber, schisandra chinensis, raw rhubarb, coix seed and patrinia. The weight parts of each component are as follows: 10 parts of red ginseng, 7 parts of prepared aconite tuber, 7 parts of schisandra chinensis, 7 parts of raw rhubarb, 13 parts of coix seed and 11 parts of patrinia.

[0042] The preparation method is the same as that in Example 1.

[0043] 1. Materials and methods

[0044] 1.1 Experimental animals

[0045] 60 SPF male healthy SD rats with a body weight of 200±20 g were purchased from Beijing Vantolliva Experimental Animal Technology Co., Ltd. (License No. SCXK (Jing) 2021-0006). The rats were bred in a barrier-level animal room, and the breeding conditions of the rats were as follows: constant temperature (22±2℃), constant humidity (45±5%), free access to tap water and food, 12 hours of light, and day-night cycle. After 1 week of adaptive feeding, the experiment was started.

[0046] 1.2 Experimental drugs

[0047] The traditional Chinese medicine composition prepared in Example 1, i.e., the gastrointestinal recovery formula, is composed of red ginseng, prepared aconite tuber, schisandra chinensis, raw rhubarb, coix seed and patrinia in a mass ratio of 9:9:6:9:12:12, and is processed into extract paste (3.25 g of crude drug) by the preparation room of Xiyuan Hospital of China Academy of Chinese Medical Sciences, and is stored in a 4℃ refrigerator. Dexamethasone tablets are produced by Guangdong Nanguo Pharmaceutical Co., Ltd. (Approval No. Guo Yao Zhu H44024618), and the specification is 0.75 mg×100 tablets.

[0048] 1.3 Experimental reagents and experimental instruments

[0049] Lipopolysaccharide (LPS, batch number: L2880-100MG) was purchased from Sigma (USA) company. Rat IL-6 ELISA kit (batch number: ERC003.96), rat TNF-a ELISA kit (batch number: ERC102a.96), rat IFN-g ELISA kit (batch number: ERC101g.96), rat IL-1b ELISA kit (batch number: ERC007.96) were all purchased from Beijing Xingboseng Biotechnology Co., Ltd. Proliferating cell nuclear antigen (PCNA) (batch number: E-AB-18205) was purchased from Wuhan Elyra Biotech Co., Ltd. HRPIgG (H+L) Rabbit-Goat IgG (batch number: Bs-0295G-HRP) was purchased from Beijing Boaosen Biotechnology Co., Ltd. TUNEL cell apoptosis detection kit (batch number: 20230801) was purchased from Yixing Biotechnology Co., Ltd. BCA protein quantification kit (batch number: WB0028), goat anti-rabbit IgG (H+L), HRP (batch number: S004) were all purchased from Beijing Tiandeyue Biotechnology Co., Ltd. Goat anti-mouse IgG (H+L), HRP (batch number: S001) was purchased from Beijing Biotechnology Co., Ltd. GAPDH mouse monoclonal antibody (batch number: YM3029) was purchased from Immunoway (USA) company. HSP90AA1 rabbit polyclonal antibody (batch number: GTX109753) was purchased from Genetex (USA) company. Caspase3 rabbit polyclonal antibody (batch number: 9662) was purchased from CST (USA) company.

[0050] Inverted microcirculation observation system (model number: GeneI-MC1) was produced by Olympus (Japan) company. Orthosteric optical microscope (model number: Eclipse E100) was produced by Nikon (Japan) company. Fluorescence microscope (model number: HS-1145) was produced by Chongqing Aote Optical Instrument Co., Ltd. Low-temperature refrigerated centrifuge (model number: Fresco21) was produced by Thermo (USA) company. Enzyme label instrument (model number: MultiSkan3) was produced by Thermo (USA) company.

[0051] 1.4 Animal grouping

[0052] After 7 days of adaptive feeding of 60 SPF healthy SD male rats, they were randomly divided into ① control group (continuous 7d 2ml 0.9% pure water gavage 1 time / d + the 8th day tail vein injection of NS (10mg / kg)), ② model group (continuous 7d 2ml 0.9% pure water gavage 1 time / d + the 8th day tail vein injection of LPS (10mg / kg) modeling), ③ dexamethasone group (continuous 7d 2ml dexamethasone suspension gavage 1 time / d + the 8th day tail vein injection of LPS (10mg / kg) modeling), ④ Weichangfugong high-dose group (continuous 7d 1.26g / ml traditional Chinese medicine 2ml gavage 1 time / d + the 8th day tail vein injection of LPS (10mg / kg) modeling), ⑤ Weichangfugong medium-dose group (continuous 7d 0.63g / ml traditional Chinese medicine 2ml gavage 1 time / d + the 8th day tail vein injection of LPS (10mg / kg) modeling), ⑥ Weichangfugong low-dose group (continuous 7d 0.315g / ml traditional Chinese medicine 2ml gavage 1 time / d + the 8th day tail vein injection of LPS (10mg / kg) modeling), a total of 6 groups, 10 rats in each group. The animal experiment program strictly follows the relevant guidelines and regulations for animal experiments, and is approved by the Animal Ethics Committee of Xiyuan Hospital of China Academy of Chinese Medical Sciences (No.: 2022XLA065-6).

[0053] 1.5 Collection of serum and intestinal tissue

[0054] 6h after modeling, rats were anesthetized with 3% sodium pentobarbital (50mg / kg), and blood was collected from the abdominal aorta of rats in each group using a disposable blood collection needle, about 5ml. Centrifuge at 3000r / min for 10min at 4℃, take the supernatant and store it in a-80℃ refrigerator for serum index detection. Explore the ileocecal region, cut off the ileum tissue about 3cm at the near ileocecal region 3-4cm with surgical scissors, soak in isotonic saline, rinse the contents and blood of the intestinal tube, divide into two parts, one is placed in 4% paraformaldehyde solution for fixation, used for intestinal histopathology, immunohistochemistry and fluorescence detection; The remaining part is stored in liquid nitrogen for related signal pathway protein level detection.

[0055] 1.6 Detection of TNF-α, IL-6 in serum and IFN-γ, IL-1β in intestinal tissue of rats in each group by ELISA method

[0056] The rat TNF-a, IL-6, IFN-gamma, IL-1beta ELISA kit is used to detect the levels of TNF-a, IL-6 in rat serum and IFN-gamma, IL-1beta in intestinal tissue. The concentrated washing solution, standard, biotinylated antibody working solution, enzyme conjugate working solution are configured into the corresponding working solution for use; 100 μL of standard working solution is added to the blank wells of the enzyme-labeled plate, 100 μL of standard sample diluent is added to the standard sample wells in proportion to the dilution, and 100 μL of sample to be tested is added to the sample wells; seal the film, shake and incubate at room temperature for 2 h; remove the sealing film, discard the liquid in the enzyme-labeled plate and pat dry on the water-absorbing paper, add 300 μL of washing solution to each well, and wash the plate 5 times; add 100 μL of corresponding biotinylated antibody working solution to each well and incubate for 1 h, wash the plate 5 times, add 100 μL of corresponding enzyme conjugate working solution to each well and incubate for 30 min, wash the plate 5 times; color development: add 100 μL of color developing substrate to each well, incubate at room temperature for 20 min in the dark; when the standard wells show a clear gradient, add 50 μL of stop solution to each well; within 5 min, detect the absorbance (OD value) of each well at 450 nm as the main wavelength and 630 nm as the reference wavelength.

[0057] 1.7 Histopathological section preparation and pathological observation

[0058] The tissue is sequentially immersed in 75%, 85%, 90%, 95%, and 100% gradient alcohol for layer-by-layer dehydration, and then immersed in xylene for a total of 30 min for fixation and transparency, followed by immersion in 65°C melted paraffin for 2 h, embedding of the tissue by embedding machine, trimming of the wax block after cooling on the freezing stage. The intestinal tissue wax block is cut into 4 μm thick sections, which are placed in a 60°C oven overnight, and then taken out for use.

[0059] Paraffin section deparaffination: sequentially place the sections in xylene I for 15 min, xylene II for 20 min for deparaffination, then place them in anhydrous ethanol I and II for 5 min each, and then rehydrate them with 75% alcohol for 5 min and tap water; hematoxylin staining: stain the sections with hematoxylin staining solution for 3-5 min, wash with tap water, then differentiate with differentiation solution, wash with tap water, and return to blue with return blue solution and running water; eosin staining: dehydrate with 85% and 95% gradient alcohol for 5 min each, then stain with eosin staining solution for 5 min; dehydration, transparency, and mounting: sequentially place the sections in anhydrous ethanol I and II for 5 min each, then dehydrate and make transparent by placing them in xylene I and II for 5 min each, and finally mount them with neutral balsam.

[0060] 1.8 Detection of intestinal tissue cell proliferation by immunohistochemical (IHC) staining

[0061] Dewaxing and rehydration. The slices were baked on a 60°C slice baker for 1 hour, then dewaxed and rehydrated; 3% H2O2 solution was added dropwise to the tissue slices for 10 min to inactivate endogenous enzymes, and the slices were washed with PBS buffer for 3 times, 5 min each time; the antigen repair solution was heated to about 95°C, then placed in the slices and heated for 30 min, and after cooling, the slices were taken out, washed with PBS buffer for 3 times, 5 min each time; normal goat serum was added dropwise for blocking, incubated at room temperature for 30 min, and the excess liquid was shaken off; the central part of the tissue was added with the primary antibody working solution, and incubated in a 4°C refrigerator for 12 h, washed with PBS buffer for 3 times, 5 min each time; the secondary antibody reaction enhancer was added dropwise, incubated at room temperature for 20 min, washed with PBS buffer for 3 times, 5 min each time; the slice was added with horseradish enzyme-labeled streptavidin working solution, incubated at room temperature for 20 min, washed with PBS buffer for 3 times, 5 min each time; DAB color developing liquid was added dropwise, incubated at room temperature for 5 min, and the color developing condition of the slice was observed under a microscope, and distilled water was added to terminate the color developing reaction, and then the slice was washed with running water; the cell nucleus was stained with hematoxylin for 10 min, dehydrated and mounted, and then observed and analyzed under a microscope.

[0062] 1.9 TUNEL staining method for detecting apoptosis of intestinal tissue cells

[0063] Dewaxing and rehydration. The slices were baked on a 60°C slice baker for 1 hour, then dewaxed and rehydrated; 3% H2O2 solution was added dropwise to the tissue slices for 10 min to inactivate endogenous enzymes, and the slices were washed with PBS buffer for 3 times, 5 min each time; the antigen repair solution was heated to about 95°C, then placed in the slices and heated for 30 min, and after cooling, the slices were taken out, washed with PBS buffer for 3 times, 5 min each time; normal goat serum was added dropwise for blocking, incubated at room temperature for 30 min, and the excess liquid was shaken off; the central part of the tissue was added with the primary antibody working solution, and incubated in a 4°C refrigerator for 12 h, washed with PBS buffer for 3 times, 5 min each time; the secondary antibody reaction enhancer was added dropwise, incubated at room temperature for 20 min, washed with PBS buffer for 3 times, 5 min each time; the slice was added with horseradish enzyme-labeled streptavidin working solution, incubated at room temperature for 20 min, washed with PBS buffer for 3 times, 5 min each time; DAB color developing liquid was added dropwise, incubated at room temperature for 5 min, and the color developing condition of the slice was observed under a microscope, and distilled water was added to terminate the color developing reaction, and then the slice was washed with running water; the cell nucleus was stained with hematoxylin for 10 min, dehydrated and mounted, and then observed and analyzed under a microscope.

[0064] 1.10 Western blot method for detecting the expression of HSP90-α and Caspase-3 in intestinal tissue

[0065] Each group selected 5 rat intestinal tissue specimens, used BCA kit for protein concentration determination; according to the need of gel electrophoresis (SDS-PAGE) loading amount, use RIPA and reducing sample buffer to adjust the protein concentration, boiling denaturation 5 min; according to the molecular weight of the target protein, according to the molecular weight of the target protein, prepare 10% separation gel and 5% concentrated gel, then pour the glue in turn, take the appropriate amount of sample and add it to the electrophoresis hole, add the pre-stained protein marker to the blank hole, and add the same amount of sample to the hole without sample to keep the gel surface balanced; the voltage of the concentrated gel is 90V, about 20min, when the sample enters the separation gel, adjust the voltage to 160V, electrophoresis to bromophenol blue just run out, then terminate electrophoresis. Membrane transfer, using wet transfer method, membrane transfer conditions: 300mA constant current, membrane transfer 2h. Blocking, primary antibody incubation: dilute the primary antibody with 5% skim milk powder-TBST, the dilution ratio of HSP90AA1 primary antibody is 1:5000, the dilution ratio of Caspase3 primary antibody is 1:2000, and the dilution ratio of GADPH mouse monoclonal antibody is 1:5000; after incubation at room temperature for 40min, wash the membrane with TBST at room temperature for 5 times, each time for 3min; dilute the secondary antibody with 5% skim milk powder-TBST, the secondary antibody is goat anti-rabbit IgG (H+L) HRP and goat anti-mouse IgG (H+L) HRP, the dilution ratio is 1:10000, incubate at room temperature for 30min, then wash the membrane with TBST for 5 times, each time for 3min; ECL reaction, film exposure, development, fixation, and image acquisition. Use Image J software to analyze the relative expression of target protein.

[0066] 1.11 Statistical analysis

[0067] All data in this experiment were described as mean ± standard deviation. Image measurement and analysis were performed using ImageJ software, data analysis was performed using statistical software SPSS 25.0, and Grapgpad Prism 8.0 was used for plotting. For comparison among multiple groups with equal variance, one-way ANOVA was used, and for comparison among multiple groups with unequal variance, non-parametric test was used. P<0.05 was considered statistically significant.

[0068] 2. Results

[0069] 2.1 Effect of Weichangfugan on pathological morphology of small intestine in sepsis rats

[0070] HE staining of rat ileal tissue was used to observe the pathological morphology. For example Figure 1The blank group ileum tissue structure was clear, intestinal villi was normal, crypt and gland structure was complete, contained rich goblet cells, mucosa layer was complete, no obvious inflammatory cell infiltration was observed. The model group mouse colon tissue structure was disorder, intestinal gland and crypt was destroyed seriously, goblet cells was absent, mucosa layer and submucosa layer had a large number of inflammatory cell infiltration; it was indicated that the model preparation was successful. Compared with the model group, the dexamethasone group ileum tissue structure was relatively complete, gland arranged more orderly, retained a large number of goblet cells, appeared visible crypt structure, inflammatory cell infiltration was obviously reduced, the improvement effect was more obvious. The gastrointestinal recovery prescription low, medium and high dose groups had different degrees of improvement of ileum injury, mainly manifested as crypt shortening alleviation, mucosa injury alleviation and submucosal inflammatory cell infiltration reduction.

[0071] 2.2 Effect of Gastrointestinal Recovery Prescription on Chiu's score of intestinal tissue of sepsis rats

[0072] The rat ileum tissue was stained by HE, and the intestinal mucosa injury was evaluated by Chiu's score of intestinal tissue. As shown in Table 1, compared with the blank group, the Chiu's score of the model group was significantly increased (P<0.01), which indicated that the model intestinal mucosa injury was serious; compared with the model group, the scores of the dexamethasone group and the gastrointestinal recovery prescription high, medium and low dose groups were decreased in different degrees, among which the scores of the dexamethasone group and the gastrointestinal recovery prescription high dose group were significantly decreased (P<0.01), and there was a statistical difference between the two groups; compared with the dexamethasone group, the gastrointestinal recovery prescription low dose group had no statistical difference (P>0.05).

[0073] Table 1 Chiu's score of intestinal tissue of rats in each group

[0074]

[0075]

[0076] Note: compared with the blank group, * P<0.05, ** P<0.01, compared with the model group, # P<0.05, ## P<0.01, compared with the dexamethasone group, && P<0.01.

[0077] 2.3 Effect of Gastrointestinal Recovery Prescription on inflammatory response of sepsis rats

[0078] The ELISA method was used to detect the levels of TNF-α, IL-6 in serum and IL-1β, IFN-γ in intestinal tissue of rats in each group. As shown in Table 2, compared with the blank group, the levels of TNF-α, IL-6 in serum and IL-1β, IFN-γ in intestinal tissue of the model group were significantly increased (P<0.01), which indicated that the model inflammatory response was serious; compared with the model group, the levels of TNF-α, IL-6 in serum and IL-1β, IFN-γ in intestinal tissue of the dexamethasone group and the gastrointestinal recovery prescription high, medium and low dose groups were decreased in different degrees, among which the levels of TNF-α, IL-6 in serum and IL-1β, IFN-γ in intestinal tissue of the dexamethasone group and the gastrointestinal recovery prescription high dose group were significantly decreased (P<0.01), and there was a statistical difference between the two groups; compared with the dexamethasone group, the gastrointestinal recovery prescription low dose group had no statistical difference (P>0.05). Figure 2Compared with the model group, the levels of TNF-α, IL-6, IL-1β and IFN-γ in the model group were increased (P<0.01). Compared with the model group, the levels of TNF-α, IL-6, IL-1β and IFN-γ in the dexamethasone group, the low, middle and high dose Weichangfuyuan groups were decreased (P<0.01), and the levels of TNF-α, IL-6, IL-1β and IFN-γ in the dexamethasone group and the high dose Weichangfuyuan group were decreased significantly (P<0.01), and there was no significant difference between the two groups (P>0.05). Compared with the model group, the levels of IL-6 and IFN-γ in the low dose Weichangfuyuan group were decreased significantly (P<0.01), and there was a significant difference between the two groups (P<0.01). Compared with the model group, the levels of IL-1β and IFN-γ in the middle dose Weichangfuyuan group were decreased significantly (P<0.01), and there was no significant difference between the two groups (P>0.05).

[0079] 2.4 Effect of Weichangfuyuan on mesenteric microcirculation in sepsis rats

[0080] The inverted microcirculation observation system was used to observe the related indexes of mesenteric microcirculation in rats. For example Figure 3Table 2, compared with the blank group, the mesenteric microartery diameter, microvenous diameter, mesenteric capillary cross network point open number of model group significantly reduced (P<0.01), mesenteric microcirculation blood flow state score significantly increased (P<0.01). Compared with the model group, the microartery diameter in dexamethasone group, gastrointestinal recovery prescription, high dose group increased in varying degrees (P<0.05 or P<0.01), with dexamethasone group and gastrointestinal recovery prescription high dose group increased significantly, and the two were statistically different (P<0.01); compared with the model group, the microartery diameter in the low dose group of gastrointestinal recovery prescription had no statistical significance (P>0.05). Compared with the model group, the microvenous diameter in dexamethasone group, gastrointestinal recovery prescription low, medium and high dose group increased in varying degrees (P<0.05 or P<0.01), with dexamethasone group and gastrointestinal recovery prescription low dose group increased significantly, and the two had no statistical difference (P>0.05); compared with the model group, the number of capillary cross network point open in dexamethasone group, gastrointestinal recovery prescription low, medium and high dose group increased in varying degrees, among which the low, medium and high dose groups of gastrointestinal recovery prescription had statistical significance (P<0.01), with dexamethasone group and gastrointestinal recovery prescription medium dose group increased significantly, and the two had no statistical difference (P>0.05); compared with the model group, the microcirculation blood flow state score in dexamethasone group, gastrointestinal recovery prescription low, medium and high dose group decreased in varying degrees (P<0.05 or P<0.01), with dexamethasone group and gastrointestinal recovery prescription medium dose group decreased significantly, and the two had no statistical difference (P>0.05).

[0081] Table 2 Comparison of the detection results of mesenteric microcirculation indexes of rats in each group n=8

[0082]

[0083] Note: compared with the normal group, *P<0.05, **P<0.01, compared with the model group, # P<0.05, ## P<0.01, compared with the dexamethasone group, & P<0.05, && P<0.01.

[0084] 2.5 Effect of gastrointestinal recovery prescription on cell proliferation in intestinal tissue of sepsis rats

[0085] The PCNA cell positive rate of intestinal tissue cells was used to evaluate the cell proliferation in intestinal tissue of rats. As Figure 4 、 Figure 5As shown in Fig. 5, compared with the blank group, the PCNA positive rate of the intestinal tissue cells of the model group was significantly reduced (P<0.01); compared with the model group, the PCNA positive rate of the dexamethasone group, the low, medium and high dose groups of Weichangfuyuan Decoction was increased to different degrees, among which the increase of the dexamethasone group and the high dose group of Weichangfuyuan Decoction was significant (P<0.01), and there was no statistical difference between the two groups.

[0086] 2.6 Effect of Weichangfuyuan Decoction on apoptosis of intestinal tissue cells of sepsis rats

[0087] TUNEL staining method was used to observe the apoptosis of intestinal tissue cells. As shown in Fig. 6, compared with the blank group, the apoptosis rate of the intestinal tissue cells of the model group was significantly increased (P<0.01); compared with the model group, the apoptosis rate of the dexamethasone group, the low, medium and high dose groups of Weichangfuyuan Decoction was significantly reduced (P<0.01), among which the reduction of the dexamethasone group and the high dose group of Weichangfuyuan Decoction was significant, and there was no statistical difference between the two groups (P>0.05). Figure 6 Figure 7

[0088] 2.7 Effect of Weichangfuyuan Decoction on expression levels of HSP90-α and Caspase-3 proteins in intestinal tissue of sepsis rats

[0089] Western blot was used to detect the expression of HSP90-α and Caspase-3 in intestinal tissue. As shown in Fig. 8, compared with the blank group, the expression levels of HSP90-α and Caspase-3 proteins of the model group were significantly increased (P<0.01); compared with the model group, the expression level of HSP90-α was reduced to different degrees in the dexamethasone group, the low, medium and high dose groups of Weichangfuyuan Decoction (P<0.05), among which the reduction of the dexamethasone group and the high dose group of Weichangfuyuan Decoction was significant, and there was statistical difference between the two groups (P<0.05); compared with the model group, the expression level of Caspase-3 protein was reduced to different degrees in the dexamethasone group, the medium and high dose groups of Weichangfuyuan Decoction (P<0.05 or P<0.01), among which the reduction of the dexamethasone group and the high dose group of Weichangfuyuan Decoction was significant, and there was no statistical difference between the two groups (P>0.01).

[0090] 3. Conclusion

[0091] ​​The research shows that the gastrointestinal dysfunction in sepsis is manifested as intestinal mucosal injury and intestinal microcirculation disorder, and its pathogenesis is closely related to inflammation, apoptosis and proliferation. The integrity of intestinal mucosal epithelial cells is conducive to the normal functioning of the intestinal barrier. During the progression of gastrointestinal dysfunction in sepsis, inflammation is one of the key pathogenic mechanisms. In the stress response of severe trauma, shock and other stress responses, due to the step-by-step amplification of inflammation, the intestinal mucosal barrier is damaged, causing the translocation of intestinal bacteria or endotoxin. Proinflammatory cytokines such as TNF-α, IL-6, IL-1β and IFN-γ can effectively initiate the anti-inflammatory process and are closely related to the prognosis of sepsis. The local inflammatory response of intestinal microcirculation can cause the activation of neutrophils, the secretion of various inflammatory mediators and reactive oxygen species, triggering a cascade of inflammatory reactions, damaging microvascular endothelial cells, and causing a large number of immune inflammatory cells to adhere to the microvascular endothelial cells. At the same time, it exacerbates the formation of microthrombosis in the microvessels by promoting platelet activation. In the later stage of sepsis, the proliferation inhibition and increased number of apoptosis of inflammation-related cells such as monocytes are one of the reasons for immune suppression. In addition, the integrity of the mechanical barrier structure of the intestinal tract also depends on the dynamic balance between the proliferation and apoptosis of intestinal epithelial cells. The apoptosis process depends on the activation of apoptosis-related protein Caspase and the regulation of HSP90-α. Caspase-3 is one of the main apoptosis factors in the apoptosis process. HSP90-α can stabilize the kinase c-Src, and the Src family kinase plays a key role in the activation of the anti-apoptotic mechanism of neutrophils.

[0092] The observation of the pathological changes of the ileum of rats in each group found that the dexamethasone group and each dose group of Weichangfuyuan Decoction could alleviate the ileum injury to a certain extent, keep the mucosal villi relatively intact, and reduce inflammatory cell infiltration. The Chiu's score of intestinal tissue in the dexamethasone group and the high, medium and low dose groups of Weichangfuyuan Decoction decreased to varying degrees. It is suggested that Weichangfuyuan Decoction can protect the intestinal mucosal barrier by promoting the repair of intestinal injury in sepsis GID rats. TNF-α, IL-6, IL-1β and IFN-γ can reflect the body's inflammatory level. The results of this experiment show that dexamethasone or Weichangfuyuan Decoction can effectively reduce the serum TNF-α, IL-6 levels of sepsis rats and the intestinal tissue IFN-γ, IL-1β levels. It is suggested that Weichangfuyuan Decoction can reduce the inflammatory response of the body and reduce the damage of LPS to the intestinal tissue of rats. The observation results of intestinal microcirculation show that Weichangfuyuan Decoction can effectively dilate the mesenteric arteriole diameter, venous diameter and capillary cross network opening number of rats, reduce the blood flow state score, and relieve the blood stasis state. The pathological essence of microcirculation disorder is blood stasis. Dahuang, Yiyiren and Baijiangcao in Weichangfuyuan Decoction can dredge the bowels and remove blood stasis, among which the effective components of anthraquinone derivatives in Dahuang, Yiyiren polysaccharide and Baijiangcao methanol in Baijiangcao can play an anti-inflammatory pharmacological role.

[0093] The immunohistochemical results showed that the three dose groups of Weichangfuyuan Decoction could increase the expression of PCNA, and the high dose group increased significantly. The TUNEL staining results showed that the three dose groups of Weichangfuyuan Decoction could reduce the apoptosis rate of intestinal tissue cells, and the high dose group reduced significantly. It is suggested that the three dose groups of Weichangfuyuan Decoction can promote the proliferation of intestinal tissue cells and inhibit the apoptosis process. The results of this experiment showed that the protein expression levels of HSP90-α and Caspase-3 in the three dose groups of Weichangfuyuan Decoction were lower than those in the model group, and the protein expression level in the high dose group was significantly reduced, indicating that Weichangfuyuan Decoction can down-regulate the protein expression of HSP90-α and Caspase-3, inhibit apoptosis and inflammatory response. The effective components of saikosaponin in red ginseng and aconite alkaloids in aconite in the composition of Weichangfuyuan Decoction have the pharmacological effect of regulating immunity. The main pathological factors of sepsis gastrointestinal dysfunction are deficiency, toxin and blood stasis. The treatment principle of Weichangfuyuan Decoction is "invigorating the spleen, restoring the function, dredging the bowels and removing blood stasis". Red ginseng and aconite can restore the function of deficiency, and Schisandra chinensis can astringe yin. The combination of various drugs can play the effects of tonifying the spleen and stomach, removing blood stasis and detoxification, and play the pharmacological effects of anti-inflammatory and immune regulation.

[0094] Conclusion: Weichangfuyuan Decoction can reduce the intestinal mucosal injury of sepsis gastrointestinal dysfunction rats, improve the mesenteric microcirculation disorder, reduce the inflammatory response of serum and intestinal tissue, inhibit the release of pro-inflammatory factors in the early stage of sepsis, reduce immune damage, inhibit intestinal tissue cell apoptosis, promote intestinal mucosal epithelial cell proliferation, and promote intestinal mucosal repair. Therefore, Weichangfuyuan Decoction has good effect on the treatment of sepsis gastrointestinal dysfunction.

[0095] 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, a number of improvements and refinements can be made, and these improvements and refinements should be considered as the protection scope of the present application.

Claims

1. A traditional Chinese medicine composition for preventing and treating sepsis gastrointestinal dysfunction, characterized in that, The raw material medicine is composed of the following components in parts by weight: red ginseng 7-10 parts, prepared aconite root 7-10 parts, Schisandra chinensis 5-7 parts, raw rhubarb 7-10 parts, coix seed 11-13 parts, and patrinia 11-13 parts.

2. The traditional Chinese medicine composition for preventing and treating sepsis gastrointestinal dysfunction according to claim 1, characterized in that, The weight parts of each component are red ginseng 9 parts, prepared aconite root 9 parts, Schisandra chinensis 6 parts, raw rhubarb 9 parts, coix seed 12 parts, and patrinia 12 parts.

3. The traditional Chinese medicine composition for preventing and treating sepsis gastrointestinal dysfunction according to claim 1, characterized in that, The weight parts of each component are red ginseng 7 parts, prepared aconite root 10 parts, Schisandra chinensis 5 parts, raw rhubarb 10 parts, coix seed 11 parts, and patrinia 13 parts.

4. The traditional Chinese medicine composition for preventing and treating sepsis gastrointestinal dysfunction according to claim 1, characterized in that, The weight parts of each component are red ginseng 10 parts, prepared aconite root 7 parts, Schisandra chinensis 7 parts, raw rhubarb 7 parts, coix seed 13 parts, and patrinia 11 parts.

5. A method for preparing a traditional Chinese medicine composition for preventing and treating sepsis gastrointestinal dysfunction, characterized in that, It comprises: The prepared aconite root, red ginseng, rhubarb, coix seed, Schisandra chinensis, and patrinia are weighed according to the proportions in the traditional Chinese medicine composition of claim 1, washed, and dried; The above decoctions are washed and placed in containers respectively for use; the prepared aconite root is first decocted for 0.5 h, and then the rhubarb, coix seed, Schisandra chinensis, and patrinia are added and decocted twice with water, the first time for 1.5 h with 8 times the amount of water, and the filtrate is collected; the second time for 1 h with 6 times the amount of water, and the filtrate is collected again; the decoctions of the two times are combined, the liquid is concentrated, cooled to room temperature, and the first decoction is obtained for standby; The red ginseng is decocted twice, the first time for 1.5 h with 8 times the amount of water, and the filtrate is collected; the second time for 1 h with 6 times the amount of water, and the filtrate is collected again; the decoctions of the two times are combined, the liquid is concentrated, cooled to room temperature, and the second decoction is obtained for standby; the first decoction and the second decoction are combined and concentrated to obtain the final product.

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