Use of chengjin tablets in preparation of drugs for regulating intestinal flora

Abstract

The application of stretch tendon tablets in the preparation of drugs for regulating intestinal flora belongs to the technical field of traditional Chinese medicines. In order to solve the technical problems that the use of the stretch tendon tablets in the prior art is limited to the treatment of tendon and bone diseases and is not associated with the regulation of intestinal flora, the present application establishes a rat intestinal flora disorder model, systematically explores the regulation of different doses of stretch tendon tablets on the structure and function of intestinal flora of the model rats, and finds that different doses of stretch tendon tablets can regulate the composition and function of intestinal flora to different degrees, improve the disorder state of the intestinal flora, and present a clear dose dependence, so that the stretch tendon tablets can be used for preparing drugs for regulating intestinal flora.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to the application of Shenjin tablets in the preparation of drugs for regulating intestinal flora. Background Technology

[0002] Shenjin Pian is a classic traditional Chinese medicine compound preparation. Its formula consists of Strychnos nux-vomica (processed with sand), safflower, frankincense (processed with vinegar), myrrh (processed with vinegar), earthworm, Stephania tetrandra, Periploca sepium, and Drynaria fortunei (processed with sand) (see the Chinese Pharmacopoeia). Current technology and clinical practice generally believe that this preparation has the effects of relaxing muscles and tendons, promoting blood circulation, removing blood stasis, reducing swelling, and relieving pain. It is suitable for arthralgia caused by cold-dampness and blood stasis obstructing the meridians, such as joint and muscle pain and swelling, aggravated by cold or exertion, numbness, and difficulty in flexion and extension, as well as diseases such as frozen shoulder, sciatica, and arthritis with the above symptoms. Currently, domestic and international research on Shenjin Pian mainly focuses on the qualitative and quantitative analysis, quality control, and safety evaluation of its constituent herbs, Strychnos nux-vomica and individual active ingredients (such as strychnine, nux vomica alkaloids, and emodin), and no systematic functional reports have been found.

[0003] With the development of microbiome research, the medical community has gradually revealed the close link between gut microbiota and various chronic diseases. In the field of bone and joint health, there is considerable evidence that gut microbiota dysbiosis can affect host immune homeostasis and nutrient absorption through the "gut-bone axis" or "gut-joint axis," thereby participating in the occurrence and development of diseases such as osteoporosis and rheumatoid arthritis. This linkage mechanism is mainly reflected in the long-range regulation of systemic inflammation levels and bone metabolic balance by microbial metabolites (such as short-chain fatty acids and lipopolysaccharides).

[0004] Currently, gut microbiota intervention strategies for musculoskeletal diseases are mostly limited to directly supplementing with exogenous probiotics and prebiotics or making dietary adjustments. While these methods have some effect, they still have limitations in terms of long-term stability, individual response variability, and safety. On the other hand, although Shenjin tablets have shown clear clinical efficacy in treating related diseases, their potential multi-target mechanism of action has not been fully elucidated. In particular, regarding its impact on the gut microbiota, current technologies have neither linked Shenjin tablets to the structural remodeling of the gut microbiota nor utilized its multi-component synergistic advantages to compensate for the shortcomings of existing microbiota intervention methods, resulting in the untapped potential of this traditional drug. Summary of the Invention

[0005] To address the technical limitations of existing technologies, such as the application of Shenjin tablets being limited to the treatment of musculoskeletal disorders and their failure to be linked to the regulation of gut microbiota, this invention establishes a rat gut microbiota disorder model and systematically explores the regulatory effects of different doses of Shenjin tablets on the structure and function of the gut microbiota in the model rats. The results show that intervention with different doses of Shenjin tablets can regulate the composition and function of gut microbiota to varying degrees, improve the disordered state of the microbiota, and exhibit a clear dose-dependent effect.

[0006] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution: The purpose of this invention is to provide the application of Shenjin tablets in the preparation of drugs for regulating intestinal flora.

[0007] In one embodiment of the present invention, the regulation of intestinal flora includes regulating the composition of intestinal flora and regulating the function of intestinal flora.

[0008] In one embodiment of the present invention, the regulation of intestinal flora composition includes increasing the abundance of genera with reduced abundance, including *Akermansia* (…). Akkermansia ), Desulfuric Vibrio spp. Desulfovibrio ) and Adlerella spp. Adlercreutzia At least one of the following.

[0009] In one embodiment of the present invention, regulating the composition of the intestinal flora includes inhibiting the overgrowth of bacteria, including *Lactobacillus* (…). Lactobacillus ).

[0010] In one embodiment of the present invention, the regulation of intestinal flora function includes regulating intestinal flora functional pathways and restoring intestinal microecological homeostasis.

[0011] The beneficial effects of this invention are: This invention establishes a rat model of intestinal flora disorder, randomly dividing experimental rats into a normal group, a model group, and low- and high-dose groups of Shenjin tablets. After continuous administration, the regulatory effect of Shenjin tablets on the structure and function of the intestinal flora in the model rats was studied. The results showed that different doses of Shenjin tablets could regulate the composition and function of the intestinal flora to varying degrees, improving the disordered state, and exhibiting a clear dose-dependent effect. Its core advantage lies in its selective enhancement... Akkermansia , Adlercreutzia The abundance of beneficial bacteria, while regulating Desulfovibrio The abundance of bacteria genus, inhibiting Lactobacillus It can promote the overgrowth of bacteria and their corresponding species, while regulating the core functional pathways of the gut microbiota and restoring gut microecological homeostasis. Attached Figure Description

[0012] Figure 1is a graph showing the results of α diversity analysis of the flora composition in fecal samples of rats in each group; Figure 2 is a graph showing the results of PCoA analysis of the flora composition in fecal samples of rats in each group; Figure 3 is a graph of the flora composition at the genus level in fecal samples of rats in each group; Figure 4 is a graph showing the analysis of differential flora at the genus level in fecal samples of rats in each group; Figure 5 is a graph of the flora composition at the species level in fecal samples of rats in each group; Figure 6 is a graph showing the analysis of differential flora at the species level in fecal samples of rats in each group; Figure 7 is a graph showing the analysis of flora functional differences in fecal samples of rats in each group. Detailed Description of the Embodiments

[0013] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below with reference to specific embodiments and the accompanying drawings of the specification. It should be noted that the embodiments mentioned below are only used to explain the present invention, but not to limit the scope of the present invention. The embodiments mentioned below are only a part of the embodiments of the present invention, not all of them. Persons skilled in the art can refer to the content of this text and appropriately improve the process parameters to achieve the objectives of the present invention. It is particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate modifications and combinations to the method and application described herein without departing from the content and scope of the present invention to realize and apply the technology of the present invention. In the field, if other technicians do not perform creative work, all the embodiments obtained by them shall fall within the protection scope of the present invention.

[0014] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents and instruments used are conventional materials, reagents and instruments in the art unless otherwise specified, all of which can be obtained through commercial channels by those skilled in the art.

[0015] The experimental animals used in the present invention are as follows: 40 4-week-old weaned male Wistar rats weighing 60-75 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., with the animal license number: SCXK (Beijing) 2021-0006. The rats were reared in a standard animal room at a temperature of 22-25°C and a humidity of 50%-60%, with a 12 h / 12 h day-night cycle, free access to food and water, and the experiment was carried out after 1 week of adaptive rearing.

[0016] The experimental drugs and reagents used in this invention are as follows: The Shenjin tablets were provided by Jilin Aodong Yanbian Pharmaceutical Co., Ltd., and were prepared into a suspension with physiological saline after being ground.

[0017] Example 1: Application of Shenjin tablets in regulating intestinal flora 1. Experimental grouping, modeling, and drug administration Healthy rats were selected and divided into 4 groups of 10 each, as follows: Group C (normal control group, no model construction or intervention), Group M (model group, intestinal flora disorder model was constructed, no intervention), Group D (low-dose intervention group, intestinal flora disorder model was constructed and low-dose target intervention agent was administered), and Group G (high-dose intervention group, intestinal flora disorder model was constructed and high-dose target intervention agent was administered).

[0018] Modeling method: After a one-week adaptation period, 40 rats were randomly divided into two groups: group C (n=10) and group D (n=30) were induced to develop the model using lincomycin hydrochloride at a dose of 6 g / kg. -1 The rats were administered the medication twice daily by gavage for three consecutive days. During the modeling period, group C was administered an equal volume of physiological saline by gavage. Successful modeling was indicated by the appearance of watery, loose stools. Twelve hours later, ten successfully modeled rats were randomly selected as the intestinal flora imbalance model group (Group M). The remaining successfully modeled rats were randomly divided into two groups (low-dose and high-dose groups), with ten rats in each group. The low-dose and high-dose groups were administered 225 mg / kg of the medication. -1 450 mg·kg -1 The rats were treated with Shenjin tablets (ground and prepared into a suspension with physiological saline) for a total of 5 days. During the intervention period, groups C and M were administered an equal volume of physiological saline by gavage. After the administration was completed, the rats were sacrificed, and the intestinal contents (>0.5 g) were collected under aseptic conditions and stored in sterile cryovials at -80°C for further analysis.

[0019] After the intervention, fecal samples were collected from rats in each group to detect the composition of the gut microbiota. Data collection and analysis were performed using the Paisenno Gene Cloud Platform. Based on the 16S rRNA gene sequencing results of the fecal samples from each group of rats, α-diversity analysis of the gut microbiota was conducted, including the calculation of the Shannon index (assessing species diversity and evenness), the Simpson index (assessing species dominance), the Pielou evenness index (Pielou_e) (assessing the evenness of species distribution), and the Allen's_H index (assessing the comprehensive characteristics of species richness and diversity). One-way ANOVA was used to compare the α-diversity indices among the groups, with P < 0.05 considered statistically significant. PCoA analysis was used to assess the differences in the overall structure of the gut microbiota among the groups of rats; species difference analysis, species-level composition difference analysis, and DiffStat analysis were used to screen for differentially expressed gut microbiota among the groups; functional difference analysis (Picrust2 and DiffStat) was used to screen for differences in functional pathways among the groups, with a focus on analyzing the regulatory effects of drug treatment on the composition and function of the gut microbiota in groups D and G.

[0020] 2. Analysis of α-diversity of gut microbiota in each group of rats This invention analyzes the α-diversity of gut microbiota in rats of each group by calculating the Simpson index, Pielou'e index, Shannon index, and Allen's H index, respectively. The results are as follows: Figure 1 As shown, compared with the normal control group (C), the four α-diversity indices of the model group (M) were significantly decreased (P<0.05). After intervention with low-dose (D) and high-dose (G) Shenjin tablets, each α-diversity index further showed a dose-dependent increasing trend, and the differences between groups were statistically significant (Simpson, P=0.024; Pielou_e, P=0.0038; Shannon, P=0.013; Allen's_H, P=0.0029). The above results indicate that Shenjin tablets can significantly improve the α-diversity of the gut microbiota, and the abundance and evenness of dominant species are restored, suggesting that it may reshape the gut microbiota structure by selectively enriching specific functional bacteria.

[0021] 3. Analysis of differences in the overall structure of the gut microbiota among the rat groups (PCoA analysis) β-diversity analysis employed principal coordinate analysis (PCoA) and was evaluated based on Bray-Curtis distance. Figure 2The results showed that PC1 and PC2 accounted for 36.6% and 15.3% of the total variation, respectively, totaling 51.9%. Groups C and M were significantly separated on the PCoA plot, indicating that lincomycin hydrochloride induced a significant change in the overall structure of the gut microbiota. After intervention with low and high doses of Shenjin tablets, the microbiota composition of groups D and G was relatively similar, still separated from group C, but showing a clear shift trend. These results demonstrate that the Shenjin tablets described in this invention can significantly alter the β-diversity structure of the gut microbiota, bringing the microbiota composition closer to that of normal rats.

[0022] 4. Analysis of differences in gut microbiota composition among different groups of rats (1) Analysis of the composition and differences of the genus level The analysis results showed significant differences in gut microbiota among different groups of rats in multiple genera (P<0.05). Genus-level composition analysis showed ( Figure 3 The gut microbiota of rats is mainly composed of Alternaria species ( Allobaculum ), Lactobacillus Bifidobacterium spp. Bifidobacterium ), Akkermansia Composed of bacteria of the genus *[[ ]*. Compared to group C, group M contains...* Lactobacillus The abundance increased significantly, indicating an over-proliferation state. Allobaculum , Bifidobacterium , Akkermansia Abundance decreased significantly. This bacterial community disorder could be reversed after intervention with Shenjin tablets.

[0023] At the genus level, differential analysis (DiffStat) was used to screen for genera with significant differences between groups. Figure 4 The results showed that the gut microbiota in group M was significantly altered compared to group C. Akkermansia , Desulfovibrio , Adlercreutzia The abundance of all samples decreased significantly. Lactobacillus Then there is excessive proliferation. Compared with group M, after the intervention of the tendon-stretching tablets, Akkermansia (between groups P=0.0064) Adlercreutzia (between groups P=0.0422) Desulfovibrio (Between groups P=0.0338) Abundance increased significantly in all groups. Lactobacillus (P=0.0013) Abundance decreased significantly, and the relative abundance of the bacterial community approached that of normal rats. These results indicate that Shenjin tablets can significantly regulate the abundance of several key bacterial genera, especially selectively increasing... Akkermansia , Adlercreutzia The abundance of beneficial bacteria, while regulating Desulfovibrio The abundance of bacteria genus, inhibiting Lactobacillus This can promote the overgrowth of bacteria and their corresponding species, thereby improving the imbalance of the intestinal microecology.

[0024] (2) Analysis of species composition and differences Species-level composition analysis showed that there were significant differences in the abundance of core bacterial species between groups M and C. Groups D and G showed targeted regulation of core bacterial species, as detailed below: Species composition analysis shows that ( Figure 5 The gut microbiota of rats is mainly composed of Bifidobacterium animalis (Bifidobacterium praecox). Bifidobacterium animalis Bifidobacterium pseudolongum ( Bifidobacterium pseudolongum Lactobacillus reuteri ( Lactobacillus reuteri Akkermania ( Akkermansia muciniphila Lactobacillus helveticus ( Lactobacillus helveticus The composition of the microbial community in group M was as follows: (e.g., M group showed abnormalities compared to group C, while the microbial community compositions of groups D and G were similar to those of group C).

[0025] At the seed level, the results of the difference analysis showed that ( Figure 6 Compared to group C, group M Akkermansia muciniphila Abundance decreased significantly. Lactobacillus reuteri Abundance increased significantly, with all inter-group differences P < 0.05. After the intervention with the stretching tablets, compared with group M, Akkermansia muciniphila Abundance increased significantly. Lactobacillus reuteri The abundance decreased significantly and then returned to normal levels. These results indicate that Shenjin tablets can precisely regulate multiple key functional bacterial species at the species level, especially... Akkermansia muciniphila and Lactobacillus reuteri .

[0026] 5. Analysis of differences in gut microbiota function The significant differences in gut microbiota functional pathways among the groups were identified through MetaCyc pathway differential analysis (DiffStat). Figure 7The results showed that after intervention with Shenjin tablets, the following pathways were affected: pyruvate fermentation to propionic acid and acetic acid (PWY-6126, P=0.0039), L-tryptophan degradation (PWY-5686, P=0.0034), adenosine nucleotide degradation (PWY-7219, P=0.0029), superoxide radical degradation (PWY-7229, P=0.0039), pyrimidine deoxyribonucleotide phosphorylation (PWY-7222, P=0.0101), and purine nucleotide degradation (PWY-722). The relative abundances of the following pathways were significantly altered (P<0.05 or P<0.01): 0, P=0.0101; pentose phosphate pathway (PWY-5100, P=0.0012); L-methionine biosynthesis pathway (PWY-7208, P=0.0033); and non-oxidative pentose phosphate pathway (NONOXIPENT-PWY, P=0.0038), approaching the levels of the normal control group. However, the anaerobic glycolysis pathway (ANAGLYCOLYSIS-PWY) showed no significant difference (P=0.0576). These results indicate that the Shenjin tablets described in this invention can significantly regulate multiple key metabolic pathways of the intestinal flora, especially short-chain fatty acid production, tryptophan metabolism, antioxidant defense, nucleotide metabolism, and glucose metabolism pathways, thereby exerting a regulatory effect on the intestinal microecology.

[0027] The main regulated pathways are core pathways for the survival, proliferation, and probiotic functions of beneficial gut bacteria. Normal expression of these pathways synergistically ensures energy supply and nutrient synthesis for beneficial bacteria, promotes the colonization and proliferation of beneficial bacteria such as Bifidobacteria and Akkermansia, and simultaneously synthesizes active metabolites such as arginine, polyamines, cladinoic acid, folic acid, and vitamins. This achieves positive regulatory effects, repairing the host intestinal mucosal barrier, regulating intestinal immunity, and supplementing micronutrients, bringing the gut microbiota's metabolic function close to normal levels. This regulatory model achieves the restoration of gut microbiota species composition at the metabolic function level, possessing targeting, specificity, and effectiveness, providing a novel and effective solution for gut microbiota regulation.

[0028] The above results indicate that rats in group M exhibited significant dysbiosis in gut microbiota structure and function. Both low and high doses of the target intervention agent, Shenjin tablets, could regulate gut microbiota composition and function to varying degrees, improving the dysbiosis, and showing a clear dose-dependent effect. Its core advantage lies in its selective enhancement... Akkermansia , Desulfovibrio , Adlercreutzia The abundance of *Isatis* genera and corresponding species, inhibiting Lactobacillus It can promote the overgrowth of bacteria and their corresponding species, while regulating the core functional pathways of the gut microbiota and restoring gut microecological homeostasis.

[0029] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of Shenjin tablets in the preparation of a drug for regulating lincomycin hydrochloride-induced antibiotic-associated intestinal flora dysbiosis, characterized in that, The regulation includes regulating the composition and function of the gut microbiota; the regulation of the gut microbiota composition includes increasing the abundance of genera with reduced abundance, including *Akermansia* (…). Akkermansia ), Desulfuric Vibrio spp. Desulfovibrio ) and Adlerella spp. Adlercreutzia At least one of the following: ; and inhibition of over-proliferating bacteria, including Lactobacillus ( Lactobacillus The regulation of gut microbiota function includes modulating gut microbiota functional pathways and restoring gut microecological homeostasis.

Images