Application of Battarian seven-ingredient pill in treatment of intestinal diseases

Abstract

The invention provides an application of a Battarian seven-ingredient pill in preparation of a medicine for improving intestinal flora imbalance. The invention further provides application of the Battalian seven-ingredient pill in preparation of a medicine for treating the viscous intestinal stabbing disease.

Description

Technical Field

[0001] The present invention relates to the field of inventions for the use of drugs for intestinal flora imbalance or "sticky intestinal irritation", and in particular to the use of Bateri Qiwei Pills in the preparation of intestinal flora imbalance or "sticky intestinal irritation". Background Art

[0002] Bateri Seven-Ingredient Pills are a traditional Mongolian formula, now included in the "Mongolian Medicine Volume, Drug Standards, Ministry of Health of the People's Republic of China" (1998 edition). This recipe has a cooling effect and is commonly used as the main ingredient for intestinal irritation and mucus-heat syndromes. It features Aconitum kusnezoffii (Aconitum carmichaelii), which relieves pain and eliminates mucus, as the main ingredient. It is supplemented with Oxytropis multiflora (Oxytropis multiflora), which eliminates musk, which eliminates mucus and stimulates the orifices, and Heiyunxiang (Heiyunxiang), which eliminates mucus and relieves pain. It also features Rubia cordifolia (Rubilai), which clears heat, stops bleeding, and stops diarrhea, as the auxiliary ingredient. Terminalia chebula (Terminalia chebula), which regulates the vital energy and detoxifies, and Vermilion vermilion (Crimson Vermilion), which clears heat, astringes, and heals wounds, as the guiding ingredients. These seven herbs, combined in the formula, offer the benefits of clearing heat and detoxifying, eliminating mucus, relieving pain, dispersing blood stasis, and stopping diarrhea.

[0003] The intestine is a vital organ for digestion and absorption in the human body, and a key barrier against the invasion of external antigens. The intestine is home to a vast bacterial community (approximately 100 trillion bacteria, 500-1000 species), which are primarily divided into three categories: Beneficial bacteria (probiotics), such as Bifidobacterium and Lactobacillus, synthesize essential vitamins (B vitamins, K, etc.) and amino acids, promote mineral absorption, participate in metabolism, inhibit pathogenic bacteria, and decompose harmful substances, making them crucial for health; Harmful bacteria, whose excessive proliferation can cause disease, produce carcinogens, and impair immune system function; and Neutral bacteria, such as Escherichia coli and Enterococcus, which are beneficial under normal circumstances, but can cause disease if they proliferate or shift uncontrollably.

[0004] Human health is closely linked to the structure of beneficial bacteria. Over a long period of evolution, the intestinal flora has formed a dynamically balanced microecological system with the host and the environment. This microecological balance is influenced by both the host and the external environment. Beneficial bacteria maintain their own stability and the health of the host by producing antibacterial substances and competing for nutrient space. This balanced state plays a fundamental role in maintaining normal nutritional, immune, and digestive functions of the host.

[0005] The intestinal microbiota is considered the body's "second genome," and its imbalance is closely associated with a variety of digestive system diseases (such as inflammatory bowel disease), metabolic diseases (such as diabetes), and autoimmune diseases. Reduced microbial diversity, compositional changes, and altered spatial distribution play a key role in regulating colonic mucosal immunity.

[0006] Intestinal dysbacteriosis refers to a condition in which factors such as age, diet, and drug abuse suppress sensitive intestinal bacteria, allowing uninhibited bacteria to multiply, leading to an imbalance in the intestinal flora. This disrupts the normal physiological composition, creating a pathological composition and causing clinical symptoms. Patients primarily experience diarrhea, abdominal pain, bloating, and bowel sounds, which may be accompanied by fever, nausea, and vomiting. The key to treating this condition lies in correcting the imbalanced intestinal microecological environment and restoring normal intestinal flora. Complications include irritable bowel syndrome, multiple organ failure, and inflammatory bowel disease.

[0007] Intestinal adhesions can cause stabbing pain. These adhesions form between the intestine and the peritoneum after abdominal surgery and can be caused by inflammation, abdominal infection, or intestinal disease.

[0008] This invention is based on metabolomics combined with intestinal flora and takes the holistic view of Mongolian medicine as the guiding ideology. It combines the macroscopic ideas of traditional Mongolian medicine with modern microscopic science and technology, and applies Bateri Seven Flavor Pills to improve intestinal flora imbalance or "sticky intestinal irritation disease". Summary of the Invention

[0009] The pharmacodynamic results of the present invention showed that Bateri Qiwei Pills can effectively improve colon shortening and weight loss in rats with "viscous intestinal irritation", and each treatment group can effectively reduce the DAI score. Histopathological results showed that each treatment group can significantly improve "viscous intestinal irritation".

[0010] The ELISA results show that the high-dose group of Batrir Qiwimei Pill significantly down-regulates TNF-α, IL-1β, IL-6, CRP, IFN-γ, DAO and MPO, and up-regulates the expression levels of IL-10 and EGF (P<0.05). The results of regulating the intestinal flora of immune function show that Batrir Qiwimei Pill can regulate the state of the intestinal flora of rats. The alpha diversity and beta diversity analysis shows that there is an obvious difference in the intestinal flora among the normal group, the model group and the Batrir Qiwimei Pill group. Further analysis of the relative abundance of the flora shows that, compared with the normal group, the relative abundance of Bacillota in the model group is significantly up-regulated, and the relative abundance of Bacteroidota is significantly down-regulated; compared with the model group, the relative abundance of Bacillota in the Batrir Qiwimei Pill group is significantly down-regulated, and the relative abundance of Bacteroidota is significantly up-regulated. At the genus level, compared with the normal group, the relative abundance of Ruminococcus in the model group is significantly up-regulated, and the relative abundance of Phocaeicola and Prevotella is significantly down-regulated; compared with the model group, the relative abundance of Ruminococcus in the Batrir Qiwimei Pill group is significantly down-regulated, and the relative abundance of Phocaeicola and Prevotella is significantly up-regulated. The results of Western blotting show that the expression of Bcl-2 is absent in the rats with “sticky intestinal pain disease”, and the NF-κB p65, IKB alpha, Caspase 3 and Bax proteins are highly expressed. After treatment with Batrir Qiwimei Pill, the expression of Bcl-2 protein in the rats with “sticky intestinal pain disease” can be significantly up-regulated, and the inflammation pathways of NF-κB p65, IKB alpha, Caspase 3 and Bax can be inhibited.

[0011] The application provides a use of Batrir Qiwimei Pill in preparation of a medicine for improving intestinal flora imbalance.

[0012] The application provides a use of Batrir Qiwimei Pill in preparation of a medicine for treating sticky intestinal pain disease.

[0013] In one of the embodiments, the use of the application is characterized in that the use is for treating diarrhea, abdominal distension, abdominal pain, nausea and vomiting caused by intestinal flora imbalance; preferably, the intestinal flora imbalance is an increase in Bacteroidota, Phocaeicola, Prevotella and / or a decrease in Bacillota and Ruminococcus; preferably, the intestinal flora imbalance is intestinal flora imbalance in patients with sticky intestinal pain disease.

[0014] In one of the embodiments, the use of the application is characterized in that the use is for treating sticky intestinal pain disease caused by intestinal flora imbalance; preferably, the use is for intestinal flora imbalance of sticky intestinal pain.

[0015] In one embodiment, the use of the present invention is characterized in that the use is to reduce the expression of TNF-α, IL-1β, IL-6, CRP, IFN-γ, DAO or MPO in patients with viscoelastic bowel disease.

[0016] In one embodiment, the use of the present invention is characterized in that the use is to increase the expression of EGF and / or IL-10 in patients with viscoelastic bowel disease.

[0017] In one embodiment, the use of the present invention is characterized in that the use is to reduce the expression of NF-κBp 65, IKB alpha and / or Caspase 3 in patients with viscoelastic bowel disease.

[0018] In one embodiment, the use of the present invention is characterized in that the use is to increase the expression of Bcl-2 in patients with viscoelastic bowel disease.

[0019] In one embodiment, the use of the present invention is characterized in that the use is to improve the intestinal flora of patients with sticky intestinal pain; preferably, the improvement of the intestinal flora of patients with sticky intestinal pain is to increase Bacteroidota, Phocaeicola, Prevotella and / or reduce Bacillota, Ruminococcus.

[0020] In one embodiment, the use of the present invention is characterized in that the dosage of the Bateri Qiwei Pills is 0.45 to 1.8 g·kg -1 As the preferred dosage of the Bat Day seven flavor ane is 0.45g kg -1 , 0.9g·kg -1 , 1.8g·kg -1 . .

[0021] Attached photos

[0022] Figure 1 : Observation of general conditions of rats in each group

[0023] Figure 2 : Optical microscopic observation of the colon tissue of rats in each group (HE staining, ×100)

[0024] Figure 3 Effects of Bateri Qiwei Pills on the levels of TNF-ɑ, IL-1β, IL-6, IL-10, CRP, EGF and IFN-γ in the serum of rats with viscoelastic intestinal disease

[0025] Figure 4 Effects of Bateri Qiwei Pills on the Contents of DAO and MPO in Colonic Tissue of Rats with Viscous Intestinal Stabbing Disease

[0026] Figure 5 : Sample dilution curve

[0027] Figure 6 :Analysis of Alpha Diversity of Intestinal Microbiota

[0028] Figure 7 :PCoA analysis of intestinal flora

[0029] Figure 8 : LEfSe analysis of intestinal flora

[0030] Figure 9 : Relative abundance of gut microbiota at the phylum and genus levels

[0031] Figure 10 :Protein band diagram

[0032] Figure 11 : Relative expression of each protein DETAILED DESCRIPTION

[0033] Example 1

[0034] 1. Experimental Materials

[0035] experimental animals

[0036] Sixty SPF male Sprague-Dawley rats, weighing (180 ± 200) g, were purchased from Changchun Yisi Laboratory Animal Technology Co., Ltd. (certificate number SCXK(Ji)2024-0001). All animals were housed in a well-ventilated experimental animal room with a temperature of 20–26°C, a relative humidity of 30%–40%, and a 12-h light–dark cycle. They were fed a standard maintenance diet according to experimental requirements. This experiment was approved by the Animal Care and Use Committee of Inner Mongolia University for Nationalities (Ethics Number: NM-LL-2023-06-15-01).

[0037] Experimental drug preparation

[0038] Bateri Qiwei Pills (Second Recipe) were purchased from the Preparation Center of the Affiliated Hospital of Inner Mongolia University for Nationalities, batch number: 20231202. According to the oral gavage dose of 0.45 g / kg for the Bateri Qiwei Pills low-dose group, 0.90 g / kg for the Bateri Qiwei Pills medium-dose group, 1.80 g / kg for the Bateri Qiwei Pills high-dose group, and 0.5 g / kg for the positive control group sulfasalazine enteric-coated tablets, Bateri Qiwei Pills and sulfasalazine enteric-coated tablets were crushed into fine powder for later use, and the fine powder was prepared into a suspension with 0.5% CMC-Na for oral gavage every day.

[0039] 2. Experimental drugs and reagents

[0040] Table 1 Experimental drugs and reagents

[0041]

[0042] 3. Experimental instruments

[0043] Table 2 Experimental instruments

[0044]

[0045] 4 Experimental methods

[0046] 4.1 Establishment of rat model

[0047] Sixty male Sprague-Dawley rats, SPF grade, weighing 180-200g, and 6 weeks old, were fed an SPF-grade diet and maintained at a room temperature of 25±0.5°C, a relative humidity of 55±5%, and a 12-hour light-dark cycle. The "viscous intestinal irritation" rat model was established by ad libitum drinking 4% DSS for 7 days in both the treatment group and the model group. The model was successfully established by the appearance of blood in the stool or diarrhea.

[0048] 4.2 Experimental Animal Grouping and Dosing

[0049] Rats were adaptively fed for one week and randomly divided into a normal group, a model group, a low-, medium-, and high-dose Bateri Qiwei Wan group, and a positive control group, with 10 rats in each group. Except for the normal group, the remaining rats were allowed to freely drink a 4% DSS aqueous solution to establish a "viscous intestinal irritation disease" rat model for 7 consecutive days, and the model was successfully established by the appearance of blood in the stool or diarrhea. Starting on the fourth day of modeling, the rats in the drug-treated groups were given oral gavage every day. The low-, medium-, and high-dose Bateri Qiwei Wan groups received doses of 0.45g / kg, 0.90g / kg, and 1.80g / kg, respectively, and the dose of sulfasalazine enteric-coated tablets was 0.5g / kg. Rats in the normal and model groups were given the same volume of normal saline once a day for 10 consecutive days.

[0050] 4.3 Observation of the disease activity index (DAI) in rats

[0051] During DSS administration, the rats' body weight and stool characteristics were observed and recorded daily. A score was also calculated based on the rats' various symptoms. DAI = (physique decline score + stool characteristics score + blood in stool score) / 3. The results are shown in Table 3.

[0052] Table 3 DAI score table

[0053]

[0054]

[0055] 4.4 Collection of Rat Serum, Urine, Colon Tissue, and Feces Samples

[0056] Serum collection: 10 days after administration, rats were fasted but not watered for 12 hours. Blood was collected from the abdominal aorta 2 hours after gavage. The blood was kept at room temperature for 0.5 hours and then centrifuged at 4°C and 4000 rpm for 15 minutes. The supernatant was aspirated, divided, and stored in a -80°C refrigerator for later use.

[0057] Urine Collection: Starting on day 4 of modeling, rats, except those in the normal and model groups, were gavage-administered with varying doses of drugs once daily. After drug administration, the rats were placed in metabolic cages, and urine was collected 24 hours after drug administration. Urine samples were centrifuged at 4000 rpm for 15 minutes at 4°C. The supernatant was aspirated, aliquoted, and stored at -80°C until further use.

[0058] Colon Collection: Using scissors disinfected with 75% ethanol, cut open the rat's abdominal cavity and remove the entire colon from the cecum to the anus. The excised intestinal segment was placed in pre-chilled PBS to clean the intestinal contents. A 0.5-1 cm section of colon was fixed in 4% paraformaldehyde. The remaining colon tissue was stored at -80°C for subsequent experiments.

[0059] Fecal collection: Using scissors and forceps disinfected with 75% ethanol, the rat's abdominal cavity was opened and the entire colon from the cecum to the anus was removed. The colonic contents were placed in sealed sterile EP tubes and stored at -80°C until further use.

[0060] 4.5 Colon Macroscopic Damage Index (CMDI)

[0061] The gross morphology of the colon was observed with the naked eye and scored according to the standard Table 4.

[0062] Table 4 CMDI score table

[0063]

[0064] 4.6 Observation of pathological changes in rat colon tissue by H&E staining

[0065] Colon tissue fixed in 4% paraformaldehyde was embedded. After embedding, the tissue was cooled to solidification and then sliced ​​into 5 μm thick sections using a microtome. The specific steps are shown in Table 5.

[0066] Table 5 Colon tissue embedding steps

[0067]

[0068] After dewaxing and rehydration, the sections were stained with H&E and then dehydrated, transparentized, and mounted. The sections were then air-dried at room temperature. The specific steps are shown in Table 6. Microscopic examination and image acquisition and analysis were performed.

[0069] Table 6 H&E staining steps for colon tissue

[0070]

[0071]

[0072] 4.7 Bartech Riqiwan Pill's Effect on the Body Weight and DAI Score of Rats with "Viscous Intestinal Pain"

[0073] During the experiment, it was observed that the rats in the normal group exhibited typical healthy conditions. They were active in behavior, sensitive to external stimuli, and had natural luster on their back hair. Their feces were granular, and their eating and drinking behaviors were regular. The model group rats induced by DSS showed systematic pathological changes after 3 days of intervention. First, they exhibited behavioral inhibition, followed by physiological abnormalities. Compared with the model group, the drug groups showed a gradient improvement effect, reversed the trend of body weight loss, and slowed down the rise of DAI score, with the high-dose group showing better results. Figure 1

[0074] 4.8 Comparison of Colon Tissue Damage

[0075] The colon mucosa of the model group rats was significantly congested and edematous, while the colon mucosa of the drug groups was significantly reduced. Compared with the normal group, the CMDI score of the model group rats increased (P < 0.05); compared with the model group, the CMDI score of the drug groups decreased (P < 0.05).

[0076] Table 2: CMDI Score Results

[0077]

[0078] 4.9 Bartech Riqiwan Pill's Effect on the Pathology of Colon Tissue in Rats with "Viscous Intestinal Pain"

[0079] HE staining results showed that the colon mucosa of the normal group rats exhibited typical healthy characteristics: the epithelial layer was intact without damage, the crypt structure was regular and sufficient (with uniform distribution of goblet cells), and there was no inflammatory cell infiltration in the submucosal layer. Compared with the normal group, the colon mucosa of the model group rats was damaged, the crypt structure was sparse, the mucosal epithelium repair rate was increased, the crypt regeneration rate was increased, and the degree of inflammatory cell infiltration was significantly reduced. Compared with the model group, the colon mucosa of the drug groups showed some recovery, with increased crypt number and reduced inflammatory cell infiltration. As shown in Figure 2 ​​

[0080] Compared with the normal group, the levels of TNF-α, IL-1β, IL-6, CRP and IFN-γ in the model group rats were significantly increased, and the levels of IL-10 and EGF were significantly decreased, which was statistically significant (P < 0.01). After treatment with various doses of Bateri Qiwei Pills, the levels of TNF-α, IL-1β, IL-6, CRP and IFN-γ were significantly decreased, and the levels of IL-10 and EGF were significantly increased. This shows that Bateri Qiwei Pills treatment can significantly inhibit the expression of TNF-α, IL-1β, IL-6, CRP and IFN-γ in the serum of rats with "viscous intestinal pain disease" and increase the levels of IL-10 and EGF in the serum of rats with "viscous intestinal pain disease". The results are shown in Table 8. Figure 3 (Note: Compared with the normal group, * P<0.05, ** P<0.01, *** P<0.01; compared with the model group, # P<0.05, ## P<0.01).

[0081] Table 8 Effects of Bateri Qiwei Pills on rats with “viscous intestinal stabbing disease”

[0082]

[0083] Note: Compared with the normal group, * P<0.05, ** P<0.01, *** P<0.01; compared with the model group, # P<0.05, ## P<0.01; “—” indicates no treatment

[0084] Effects of Bateri Qiwei Pills on DAO and MPO Contents in Colonic Tissue of Rats with Viscous Intestinal Stabbing Disease

[0085] Compared with the normal group, the levels of DAO and MPO in the colon tissue of the rats in the model group were significantly increased. After treatment with various doses of Bateri Qiwei Pills, the levels of DAO and MPO in the colon tissue of the rats were significantly reduced. The above results show that Bateri Qiwei Pills treatment can inhibit the production of DAO and MPO, thereby regulating the oxidative stress response. The results are shown in Tables 9 and 10. Figure 4 (Note: Compared with the normal group, *P<0.05, **P<0.01, ***P<0.01; compared with the model group, #P<0.05, ##P<0.01).

[0086] Table 9 Effects of Bateri Qiwei Pills on DAO in rats with “viscous intestinal stabbing disease”

[0087]

[0088] Note: Compared with the normal group, * P<0.05, ** P<0.01, *** P<0.01; compared with the model group, # P<0.05, ## P<0.01; “—” indicates no treatment

[0089] Table 10 Effects of Bateri Qiwei Pills on MPO in rats with “viscous intestinal stabbing disease”

[0090]

[0091] Note: Compared with the normal group, * P<0.05, ** P<0.01, *** P<0.01; compared with the model group, # P<0.05, ## P<0.01; “—” indicates no treatment

[0092] The "viscous intestinal irritation disease" model of the present invention is characterized by the high consistency of its pathological characteristics with human diseases, the repeatability of the modeling method and the perfection of the evaluation system.

[0093] In the present invention, the immune imbalance of "sticky intestinal irritation" is manifested as overactivation of proinflammatory factors (TNF-α, IL-1β, IL-6, IFN-γ) and inhibition of anti-inflammatory factors (IL-10). In addition, oxidative stress is also one of the important mechanisms of the pathogenesis of "sticky intestinal irritation". MPO is an enzyme present in neutrophils, which can catalyze the oxidation reaction of substrates such as chloride ions and hydrogen peroxide to produce oxidative substances, and participate in inflammatory reactions and immune activity. During the inflammatory response, the activity of MPO increases, causing oxidative stress and tissue damage. Bateri Qiwei Pills can precisely intervene in the key targets of "sticky intestinal irritation" (such as TNF-α, IL-1β, IL-6, IFN-γ, MPO), while repairing the intestinal mucosal barrier (such as EGF, DAO).

[0094] In summary, Bateri Qiwei Pills can significantly improve the clinical symptoms of rats with "viscous intestinal irritation" and play a therapeutic role in "viscous intestinal irritation" by regulating inflammatory factors and oxidative stress indicators.

[0095] The histopathological sections of the present invention show that the crypt structure of the colon tissue of rats with "viscous intestinal irritation" is severely damaged, the goblet cells are greatly reduced, and there is a large amount of inflammatory cell infiltration; after treatment with low, medium and high doses of sulfasalazine tablets and Bateri Qiwei Pills, the inflammatory condition of the colon tissue of rats with "viscous intestinal irritation" is significantly improved.

[0096] Example 2 Metagenomic Observation of the Effect of Bateri Qiwei Pills on the Intestinal Microflora of Rats with "Viscous Intestinal Stabbing Disease"

[0097] The intestinal flora, a vital component of the human digestive system, is composed of approximately 100 trillion microorganisms, including probiotics, opportunistic pathogens, and harmful bacteria. Under normal conditions, probiotics maintain intestinal health by forming a biological barrier, synthesizing vitamins, and regulating immunity, while harmful bacteria are strictly restricted. However, when factors such as an unbalanced diet, overuse of antibiotics, or infection with pathogens disrupt this microecological balance, harmful bacteria proliferate and secrete toxins, directly irritating the intestinal mucosa and triggering intestinal pain.

[0098] 1. Experimental Materials

[0099] 1.1 Experimental Animals

[0100] The experimental animals were the same as in Example 1.

[0101] 1.2 Experimental reagents and instruments

[0102] Table 11 Experimental reagents and instruments

[0103]

[0104] 1.3 Database and Software

[0105] Table 12 Database and software

[0106]

[0107]

[0108] 2 Experimental methods

[0109] 2.1 Sample collection and total DNA extraction

[0110] All samples were stored at -80°C until use. Microbial genomic DNA was extracted from rat intestinal fecal samples according to the instructions provided by the kit, and the DNA quality and concentration were tested.

[0111] 2.2 Library Construction and Sequencing

[0112] DNA that meets the requirements is sheared, adjusting the time based on the target fragment length (200-500bp). The sheared products are purified and recovered using the TruSeq library construction kit magnetic beads, and the fragmented products are then analyzed. Genomic library construction is completed through end repair, adapter ligation, index PCR amplification, and purification. The library is then quantified, and paired-end sequencing is performed according to standard protocols.

[0113] 2.3 Bioinformatics Analysis

[0114] Using bioinformatics methods, we conduct in-depth analysis of the pre-processed data, including species diversity analysis, community structure analysis, gene function annotation, etc. The analysis results are then visualized.

[0115] 2.4 Experimental Results

[0116] Alpha Diversity Analysis

[0117] like Figure 5 As shown, a rarefaction curve is drawn to evaluate the sequencing depth of each sample. Each curve represents a group. The curves of each group tend to be flat, indicating that the sequencing results are sufficient to reflect the diversity contained in the current sample and can be used for further analysis. Alpha diversity is measured by the following indices: the observed species index represents the richness of the community, and the Shannon and Simpson indices represent the diversity. Figure 6 As shown in the data, the diversity of intestinal bacteria in the model group was significantly lower than that in the normal group, while that in the Bateri Qiwei Pills group was significantly higher than that in the model group, indicating that Bateri Qiwei Pills intervention can change the intestinal bacteria diversity and flora richness of rats with "viscous intestinal pricking disease".

[0118] Beta diversity analysis

[0119] like Figure 7 As shown in the PCoA analysis, the first and second principal components explained 38.75% and 24.3% of the model, respectively, indicating strong explanatory and predictive capabilities. The model group showed a significant difference from the normal group in the PCoA analysis, indicating that the intestinal flora of the model rats had changed significantly compared to the normal group. The Bateri Qiwei Pill-treated group showed a similar pattern to the normal group, indicating that the intestinal flora structure of the rats gradually returned to that of the normal group after treatment.

[0120] LEfSe analysis

[0121] like Figure 8As shown in the figure, this experiment shows species with LDA values ​​greater than 3. Compared with the model group, the dominant species in the normal group included g_Duncaniella, g_Muribaculum, f_Muribaculaceae, g_Xylanibacter, g_Prevotella, f_Pr evotellaceae, o_Bacteroidales, c_Bacteroidia, and p_Bacteroidota, while the dominant species in the model group included p_Ba cillota, c_Clostridia, o_Eubacteriales, o_Lachnospirales, f_Lachnospiraceae, and f_Oscillospirac eae, p_Bacteria_unclassified, g_Lachnospiraceae_unclassified, g_Acetatifactor, s_uncultured_Acetatifactor_sp., g_Oscillibacter, s_Bacteroides_cellulosilyticus, o_Campylobacterales, p_Campylobacterota, c_Epsilonproteobacteria, and f_Helicbacteraceae. Compared with the model group, the dominant bacterial species in the Bateri Qiwei Pill group included g_Phocaeicola, g_Lactobacillus, s_Lactobacillus_johnsonii, g_Muribaculum, f_Tannerellaceae, g_Parabacteroides, s_Phocaeicolaunclassified, s_uncultured_M uribaculum_sp., and s_Phocaeicola_vulgatus. In summary, the dominant bacterial species in the Bateri Qiwei Pills and normal groups were similar in composition, indicating that Bateri Qiwei Pills could change the structural composition of the intestinal flora in rats with "viscous intestinal irritation" and inhibit the proliferation of certain bacteria.

[0122] like Figure 9As shown in the figure, the intestinal flora of rats in each group was mainly composed of Candidatus_Saccharibacteria, Chytridiomycota, Euryarchaeota, Synergistota, Chloroflexota, Fibrobacterota, Myxococcota, Elusimicrobiota, Spirochaetota, Fusobacteriota, MycoplasmatotaThermodesulfobacteriota, Actinomycetota, Verrucomicrobiota, Candidatus_Melainabacteria, Campylobacterota, Pseudomonadota, Uroviricota, Bacillota, and Bacteroidota at the phylum level. Compared with the normal group, the relative abundance of Bacillota increased in the model group, while the relative abundance of Bacteroidota decreased in the model group; whereas compared with the model group, the relative abundance of Bacillota decreased in the Bate Ri Qiwei Wan group, while the relative abundance of Bacteroidota increased in the Bate Ri Qiwei Wan group. At the genus level, the main components were Lachnoclostridium, Marseilla, Palleniella, Duncaniella, Oscillibacter, Pseudoflavonifractor, Ligilactobacillus, Eubacterium, Segatella, Muribaculum, Helicobacter, Lactobacillus, Alistipes, Parabacteroides, Roseburia, Clostridium, Ruminococcus, Phocaeicola, Bacteroides, and Prevotella. Compared with the normal group, the relative abundance of Ruminococcus increased in the model group, while the relative abundances of Phocaeicola and Prevotella decreased. Compared with the model group, the relative abundance of Ruminococcus decreased in the Bate Ri Qiwei Wan group, while the relative abundances of Phocaeicola and Prevotella increased in the Bate Ri Qiwei Wan group.

[0123] Sequencing results showed that Bateri Qiwei Pills significantly restored the microbial structure in mice. PCoA analysis of the gut microbiota in the normal, model, and Bateri Qiwei Pill groups revealed significant differences between the model group and the normal group. However, after Bateri Qiwei Pills treatment, the gut microbiota in the model group was significantly altered and approached that of the normal group, as confirmed by species composition analysis and LEfSe analysis.

[0124] Example 3 Effect of Bateri Qiwei Pills on Protein Expression in Rats with Viscous Intestinal Stabbing Disease

[0125] NF-κB p65, a core regulator of the inflammatory response, plays a key role in the development of "sticky bowel disease." When inactivated, it binds to the inhibitory protein IκBα and remains in the cytoplasm. Inflammatory stimuli activate the IKK complex to phosphorylate IκBα and promote its degradation, releasing p65 into the nucleus and initiating the transcription of proinflammatory cytokines (such as TNF-α and IL-1β) and chemokines, exacerbating the intestinal inflammatory cascade. Caspase 3, an apoptosis executioner, cleaves gasdermin D during intestinal epithelial cell apoptosis, releasing proinflammatory mediators such as IL-1β. Its activity is regulated by NF-κB and is directly linked to intestinal mucosal barrier damage. The dynamic balance between Bcl-2 and Bax, members of the Bcl-2 family, determines cell survival or apoptosis: Bcl-2 exerts an anti-apoptotic effect by inhibiting mitochondrial release of cytochrome C, while Bax promotes apoptotic signaling. In viscous bowel disease, pro-inflammatory cytokines (such as TNF-α) upregulate Bax expression by activating the JNK pathway, while simultaneously inhibiting Bcl-2. This leads to an imbalance in the Bax / Bcl-2 ratio, triggering excessive apoptosis of intestinal epithelial cells and disrupting the integrity of the intestinal barrier. This vicious cycle of NF-κB-mediated inflammation and dysregulated apoptosis is a key pathological mechanism for the continued progression of enteritis.

[0126] This part of the study analyzed and compared the expression changes of related proteins NF-κB p65, IKB alpha, Caspase 3, Bcl-2, and Bax through WB experiments, further revealing the effect of Bateri Qiwei Pills on the immune response of the rat model of "viscous intestinal irritation".

[0127] 1. Experimental Materials

[0128] 1.1 Experimental Reagents

[0129] Table 13 Experimental reagents

[0130]

[0131]

[0132] 1.2 Experimental instruments

[0133] Multifunctional gel imaging system (imported) model FUSION FX7 company VILBE

[0134] 1.3 Materials and Methods

[0135] Tissue protein extraction

[0136] Weigh a certain amount of colon tissue, grind it into a powder in a sterile mortar with liquid nitrogen, and place it in an EP tube. Add RIPA lysis buffer (10 times the volume of the tissue) and PMSF (100:1). Ultrasonicate at low temperature for 5 minutes and lyse on ice for half an hour. Centrifuge the resulting tissue homogenate at 12,000 rpm and 4°C for 5 minutes. Collect the supernatant to obtain the total protein solution, aliquot, and store at -80°C.

[0137] The protein concentration of the total protein solution was determined using BCA assay. The samples were diluted to a uniform concentration with lysis buffer, and then 5× protein loading buffer was added. The samples were boiled in a 100°C water bath for 10 min to denature the protein. The samples were stored at −20°C and thawed on ice before use.

[0138] SDS-PAGE electrophoresis

[0139] (1) Cleaning the glass plate

[0140] (2) Gel preparation and sample loading: Prepare the separating gel and stacking gel according to the gel kit instructions. Pour the gel into the gel mold and remove the gel casting apparatus after solidification. Load 30 μg of sample according to the protein concentration determination results.

[0141] (3) Electrophoresis: Add sufficient electrophoresis solution to the electrophoresis tank and start electrophoresis. Run at 80V for 30 minutes, then 120V for 60-70 minutes until the bromophenol blue is approximately 1 cm from the bottom. Terminate electrophoresis and proceed to transfer to the membrane.

[0142] 1.5 Transfer

[0143] (1) Cut the PVDF membrane into appropriate size and activate it with methanol for 2 minutes before use.

[0144] (2) Soak the transfer sponge, transfer filter paper and activated PVDF membrane in transfer buffer.

[0145] (3) Place the sponge / filter paper / gel / film on the black side of the transfer clip. Place the sponge and filter paper on the other side. Close the clip and place it in the transfer tank.

[0146] (4) Add sufficient transfer buffer, place on ice, and begin transfer. Transfer conditions: 280 mA constant current, 60 min.

[0147] immune response

[0148] (1) Blocking: Place the PVDF membrane after transfer in an incubation box with 5% skim milk powder at 4°C overnight.

[0149] (2) Cleaning: After pouring the milk powder, clean with TBST solution three times, each time for 10 minutes.

[0150] (3) Primary antibody incubation: The primary antibody was diluted with 5% skim milk powder according to the following ratios: β-actin (1:5000), GAPDH (1:6000), NF-κBp65 (1:500), IKB alpha (1:500), Caspase 3 (1:500), Bcl-2 (1:500), and Bax (1:500). The cells were placed in an incubation box and shaken at room temperature for 2 hours.

[0151] (4) Secondary antibody incubation: Recover the primary antibody and wash with TBST for 15 min × 3 times. Dilute the secondary antibody with 5% skim milk powder at a ratio of 1:10,000, place in an incubation box, and shake at room temperature for 2 hours.

[0152] (5) Color development: Recover the secondary antibody, wash with TBST for 10 min × 3 times, take it out and place it on the partition of the colorimeter, add ECL luminescent solution prepared in a 1:1 (A, B) ratio, expose and develop, and save the image.

[0153] Results and Analysis

[0154] Image J software was used to analyze the grayscale values ​​of the bands.

[0155] Experimental results

[0156] The experimental results showed that compared with the normal control group, the levels of NF-κBp65, IKB alpha, Caspase 3, and Bax proteins in the model group were significantly increased (P<0.001), and the level of Bcl-2 protein was significantly decreased; while the intervention of Bateri Qiwei Pills could significantly upregulate the expression levels of NF-κBp65, IKB alpha, Caspase 3, and Bax, and significantly reduce the expression levels of Bcl-2 (P<0.05). Figure 10 (Note: A normal group; B model group; C positive group; D Bateri Qiwei Wan low-dose group; E Bateri Qiwei Wan medium-dose group; F Bateri Qiwei Wan high-dose group) Figure 11 (Note: Compared with the normal group, * P<0.05, ** P<0.01, *** P<0.01; compared with the model group, # P<0.05, ## P<0.01, ### P<0.01).

[0157] Western blot results showed that the protein expression levels of NF-κB p65, IKB alpha and Caspase 3 were significantly up-regulated in rats with "sticky intestinal pain disease", while the expression levels were significantly decreased after the intervention of Bate Ri Qiwei Pills, suggesting that the drug may regulate inflammatory response by inhibiting the activation of NF-κB signaling pathway. At the same time, the expression of Bcl-2 protein decreased and the expression of Bax protein increased in the colon tissue of model group rats, and this imbalance of Bcl-2 / Bax ratio may be the result of pro-inflammatory factors (such as TNF-α) inducing excessive apoptosis of intestinal epithelial cells through JNK pathway under inflammatory conditions. After the treatment of Bate Ri Qiwei Pills, the protein expression trend of Bcl-2 and Bax was reversed, indicating that it may improve the status of colon tissue by the following mechanisms. On the one hand, the active ingredients such as total alkaloids of Aconitum leaves in the drug can inhibit the nuclear translocation of NF-κB p65, reduce the release of pro-inflammatory factors, and thus reduce the damage of inflammatory cascade reaction to intestinal mucosa; on the other hand, the drug may regulate the mitochondrial apoptosis pathway, restore the balance of Bcl-2 / Bax, and inhibit Caspase 3-mediated apoptosis, to treat "sticky intestinal pain disease".

Claims

1. Use of Bateri Qiwei Pills in the preparation of drugs for improving intestinal flora imbalance.

2. Use of Bateri Qiwei Pills in the preparation of medicines for treating sticky intestinal pain.

3. The use according to claim 1, characterized in that: The use is to treat diarrhea, abdominal distension, abdominal pain, nausea and vomiting caused by intestinal flora imbalance; preferably, the improvement of intestinal flora imbalance is to increase Bacteroidota, Phocaeicola, Prevotella and / or reduce Bacillota, Ruminococcus; preferably, the intestinal flora imbalance is intestinal flora imbalance in patients with sticky intestinal irritation.

4. The use according to claim 2, characterized in that The use is to treat sticky intestinal stinging caused by intestinal flora imbalance; as a preferred use, the use is sticky intestinal stinging caused by intestinal flora imbalance.

5. The use according to claim 2, characterized in that The use is to reduce the expression of TNF-α, IL-1β, IL-6, CRP, IFN-γ, DAO or MPO in patients with viscous intestinal pain.

6. The use according to claim 2, characterized in that The use is to increase the expression of EGF and / or IL-10 in patients with viscous intestinal pain.

7. The use according to claim 2, characterized in that The use is to reduce the expression of NF-κBp65, IKB alpha and / or Caspase 3 in patients with viscous intestinal pain.

8. The use according to claim 2, characterized in that The use is to increase the expression of Bcl-2 in patients with viscous intestinal pain.

9. The use according to claims 3-8, characterized in that The use is to improve the intestinal flora of patients with sticky intestinal pain; preferably, the improvement of the intestinal flora of patients with sticky intestinal pain is to increase Bacteroidota, Phocaeicola, Prevotella and / or reduce Bacillota, Ruminococcus.

10. The use according to claims 1 to 9, characterized in that: The dosage of the Bateri Qiwei Pills is 0.45-1.8 g·kg -1 As the preferred dosage of the Bat Day seven flavor ane is 0.45g kg -1 , 0.9g·kg -1 , 1.8g·kg -1 .

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