Application of Mishitong capsule in the preparation of drugs for treating intestinal flora disturbance caused by kidney stones

Abstract

The present invention belongs to the field of medical technology, and specifically relates to the use of Mishitong capsule in the preparation of drugs for intestinal flora disorder caused by kidney stones; the present invention discusses the diversity, richness and uniformity of intestinal flora through Alpha diversity analysis, hierarchical clustering analysis, PCoA analysis, community column chart (phylum and genus level) analysis, species Venn diagram analysis, heat map analysis, PCA analysis, etc. The results show that Mishitong capsule can regulate the intestinal flora of mice by increasing the proportion of Firmicutes and Proteobacteria, effectively improve the diversity, richness and uniformity of intestinal flora, improve the structure of intestinal flora, and maintain the balance of intestinal flora. The discovery of this new use provides a new method and favorable basis for the treatment of intestinal flora disorder.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to application of Mishitong capsule in preparing medicine for treating or regulating intestinal flora disorder. Background Art

[0002] As one of the most important organs in the human body, the kidney plays a role in regulating and integrating the physiological functions of the whole body. It is the most complex endocrine organ in the body, responsible for filtering impurities in the blood, maintaining the balance of body fluids and electrolytes, and finally producing urine, which is excreted from the body through the urethra. The incidence of kidney stones worldwide is 10% to 15%, and the prevalence in the Chinese population ranges from 1.61% to 20.45%. It is worth noting that there is a significant gender difference in the prevalence: the prevalence of kidney stones in men and women in my country is approximately 6.5% and 5.1%, respectively. At the same time, the number of underage patients has also shown an increasing trend year by year, with an annual growth rate of approximately 4% to 6%. The formation of kidney stones is a complex process that involves the interaction of multiple factors.

[0003] Western medicine often uses lithotripsy or surgical stone removal to treat kidney stones. This method is difficult to cure, prone to recurrence, and can easily damage kidney tissue during the process. Traditional Chinese medicine believes that stones are a type of "gonococcal disease" or "stone stranguria" in traditional Chinese medicine. When patients are sick, they will have symptoms such as "umbilical and abdominal pain", "difficult urination", and "sand and stone in the urine". There have been many cases of traditional Chinese medicine treating kidney stones in the urinary system in history, which provide a solid foundation for the use of Chinese patent medicines to treat kidney stones. Traditional Chinese medicine has more advantages than Western medicine in treating stones with a diameter of less than 1cm and mild urinary tract obstruction. It can reduce the damage to the kidneys caused by clinical surgery and can protect kidney function to a certain extent. In general, traditional Chinese medicine has unique advantages in treating diseases related to kidney stones.

[0004] Bishitong Capsule is a pure Chinese medicine preparation. Its main ingredients are oak leaf dry extract and talcum powder. It can clear away heat and dampness, promote qi and remove blood stasis. Because its capsule disintegration time is short, it is easy to absorb and has high bioavailability. It is often used for kidney stones of qi stagnation and blood stasis type and damp-heat type, and is suitable for patients with stone diameter less than 1cm.

[0005] The intestinal flora has a complex symbiotic relationship with the host, regulating the host's multiple metabolic pathways, participating in the host's physiological and biochemical activities, and thus affecting the host's health. Under normal circumstances, the intestinal flora is in a dynamically balanced ecological environment. However, when the human body suffers from kidney stones or is under stress, antibiotics, or foreign pathogens, it often leads to abnormal intestinal flora, which is manifested as abnormal changes in the type, quantity, and proportion of the intestinal flora.

[0006] Most of the existing microbial regulators are live bacteria preparations such as bifidobacteria and lactobacilli, which have problems such as difficulty in ensuring the number of live bacteria and poor stability. It has been reported in the literature that the effective ingredients of traditional Chinese medicine can support the growth of beneficial intestinal bacteria and inhibit the growth of harmful bacteria, and can be used to regulate the structure of intestinal flora. However, there are no reports on the research of Mishitong Capsules in regulating intestinal flora. Summary of the invention

[0007] The purpose of the present invention is to provide a new use of the Mishitong capsule, specifically, the use of the Mishitong capsule in the preparation of a medicine for treating or regulating intestinal flora disorders.

[0008] Furthermore, the intestinal flora disorder refers to the intestinal flora disorder caused by kidney stones.

[0009] Furthermore, the Mishitong capsule is used to improve the diversity, richness and uniformity of intestinal flora.

[0010] Advantages and beneficial effects of the present invention: The present invention has proved through a large number of experiments that Mishitong Capsules can effectively improve the diversity, richness and uniformity of intestinal flora, improve the structure of intestinal flora, and maintain the balance of intestinal flora. Therefore, it can be used in the preparation of drugs for regulating intestinal flora disorders, which can not only treat kidney stones, but also improve intestinal flora disorders caused by kidney stones. The discovery of this new use provides a new method and favorable basis for the treatment of intestinal flora disorders, and also broadens the application scope of Mishitong Capsules. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is Alpha diversity analysis;

[0012] Figure 2 It is hierarchical cluster analysis;

[0013] Figure 3 is PCoA analysis;

[0014] Figure 4 is the community column chart analysis (phylum level);

[0015] Figure 5 is the community column chart analysis (genus level);

[0016] Figure 6 It is the species Venn diagram analysis;

[0017] Figure 7 It is a heat map analysis;

[0018] Figure 8 is the PCA analysis. DETAILED DESCRIPTION

[0019] In order to more clearly illustrate the technical solution of the present invention, the present invention is further described below through specific embodiments, but these embodiments are only for illustration rather than limitation of the present invention.

[0020] 1.1 Test materials

[0021] Mishitong capsule (Jilin Aodong Yanbian Pharmaceutical Co., Ltd.); Animals: SPF-grade healthy male mice, purchased from Changchun Yisi Animal Technology Co., Ltd. AxyPrep DNA gel extraction kit (Axygen, USA); FastPfu polymerase (Beijing Quanshijin Biotechnology Co., Ltd., China); 0.2% agarose gel (Biowest, Spain).

[0022] 1.2 Test methods

[0023] 1.2.1 Kidney stone mouse model and drug administration

[0024] SPF male mice were selected and randomly divided into 5 groups after adaptive feeding, namely Control, Model, T1, T2 and T3, with 10 mice in each group. The model, T1, T2 and T3 groups were gavaged with stone-inducing solution (0.75% ethylene glycol + 0.75% ammonium chloride) at a dose of 3 mL / d for 2 weeks to establish the kidney stone model, and the Control group was allowed to drink water freely for 2 weeks. T1 group; T2 group was gavaged with low and high doses of 225 mg / kg and 450 mg / kg of Mishitong capsule for 2 weeks after 1 week of modeling, and T3 was given 5g / kg of Shenshitong granules (Jilin Wantong Pharmaceutical Group Meihe Pharmaceutical Co., Ltd.) for 2 weeks after 1 week of modeling, and the Control group and Model group were allowed to drink water freely for 2 weeks. During the experiment, the mental state, diet and drinking water status of each group of mice, as well as the hair growth status and color changes were observed, and the weight of each group of mice was weighed and recorded in real time during the experiment.

[0025] 1.2.2 Collection of mouse feces

[0026] Collect fresh feces from mice before dissection. Use a 1.5 mL sterile EP tube to directly collect fresh feces from mice. Wear sterile gloves throughout the operation to avoid contamination of the sample. Collect no less than 3 g of fresh mouse feces for each sample and store it in a sterile feces box.

[0027] 1.2.3 16S rRNA gene amplification

[0028] Microbial DNA was extracted from stool samples using the EZNA Soil DNA Kit genomic DNA extraction kit. The V1-V9 region of the bacterial 16S ribosomal RNA gene was amplified by PCR (95 °C for 2 min, followed by 27 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 60 s, with a final extension at 72 °C for 5 min). Primers 27F5'-AGRGTTYGATYMTGGCTCAG-3' and 1492R5'-RGYTACCTTGTTACGACTT-3' were used, with the barcode being an eight-base sequence unique to each sample. PCR reactions were performed in triplicate 20 μL mixtures containing 4 μL Fast Pfu buffer (5×), 2 μL (2.5 mM) dNTPs, 0.8 μL (5 μM) forward and reverse primers, 0.4 μL Fast Pfu polymerase, and 10 ng template DNA. Amplicons were extracted from 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit according to the manufacturer's instructions.

[0029] Contamination Procedure to Control False-Positive PCR Results In the 16 S full-length rRNA gene amplicon experiment, positive (E. coli isolate) and negative (ddH20) controls were introduced to ensure the accuracy of the results. The DNA concentration of the negative control was too low to construct the NGS sequencing library. Instead, the positive control was sequenced using the Sanger sequencing method.

[0030] 1.2.4 Library construction and sequencing

[0031] SMRTbell libraries were prepared from amplified DNA by blunt ligation according to the manufacturer's instructions; SequencingKiT2.0 chemistry was used in a dedicated PacBioSequelII8M Purified SMRTbell libraries from Zymo and HMP mock colonies were sequenced on cells; PacBioSequelII Purified SMRTbell libraries from mixed and barcoded samples were sequenced on cells; all amplicon sequencing was performed by Shanghai Brose Biotechnology Co., Ltd. Amplified fragments were pooled in equal amounts and subjected to paired-end 2 × 250 bp sequencing using the Illlumina MiSeq platform and MiSeq Reagent Kit v3.

[0032] 1.2.5 Analysis of sequencing data

[0033] PacBio raw reads were processed using SMRT Link data sequencing software to obtain circular consensus sequences (CCS). The minimum number of passes was set to 3 and the minimum prediction accuracy was set to 0.99 to determine the quality of CCS. The raw reads were processed by SMRTPortal, and only sequences with a length greater than 2500 bp and a certain quality standard were retained. Abnormal sequences containing 10 consecutive identical bases (which may be caused by sequencing errors or other reasons) were filtered out and further removed by removing barcodes, primer sequences, chimeras, and sequences. UPARSE was operated to set a similarity threshold (97%) to cluster the sequences and obtain different OTUs. UCHIME was used to identify and remove chimeric sequences (which may interfere with the analysis of community structure). Using the silva (SSU132) 16SrRNA database as a reference database, each 16SrRNA gene sequence was classified to find its phylogenetic position.

[0034] 2. Test results

[0035] 2.1 Comparison of body weight of mice in each group

[0036] The comparison of the weight of mice in each group is shown in Table 1-1; the comparison of diet and water is shown in Table 1-2. Experimental observations found that the weight of mice in the Model group decreased compared with the Control group during the initial kidney stone modeling process, and the mice were listless and their hair was drooping. After taking Mishitong Capsules, the condition of the mice improved and the weight loss trend was slow. This is because kidney stones cause mice to lose their appetite, which is mainly due to the loss of appetite caused by the pain and discomfort caused by kidney stones. Since the experiment was modeled by gavage, the diet and water intake of the model group was relatively small during the modeling period, and gradually increased in the later period. The final average diet and water intake was relatively lower than that of the blank group.

[0037] Table 1-1 Comparison of body weight (g) of mice in each group

[0038] Control Model T3 T1 T2 1d 21.53±0.91 20.85±0.87 20.68±0.74 21.18±0.97 19.04±0.96 6d 25.43±0.92 22.29±1.20 22.97±1.22 23.9±2.07 21.59±1.70 11d 26.4±1.04 24.09±1.42 24.38±1.10 26.09±1.72 23.74±1.61 16d 27.86±1.43 25.17±1.41 26.67±0.94 27.36±2.37 25.83±2.01 21d 30.26±1.56 27.48±1.76 28.86±1.08 29.22±2.36 28.17±2.28 28d 31.86±1.61 29.14±1.89 30.23±1.26 30.12±2.33 30.02±2.41

[0039] Table 1-2 Comparison of diet and drinking water in each group of mice

[0040] Control Model T3 T1 T2 Diet(g) 55.18 48.53 46.82 52.89 51.62 Drinking(mL) 59.26 54.86 51.03 48.76 58.03

[0041] 2.2 Alpha Diversity Analysis

[0042] Depend on Figure 1It can be seen that the Chao1 index of the T2 and T3 groups was higher than that of the Model group. The Observed species index and Shannon index of the T2 and T3 groups were higher than those of the Model group, among which the Observed species index had a significant difference (P < 0.05), and the diversity of bacterial species increased. Compared with the Model group, the Pielou's evenness index of the T2 and T3 groups increased significantly, and the species distribution was more uniform.

[0043] 2.3 Bate diversity analysis

[0044] 2.3.1 Hierarchical cluster analysis

[0045] Bate diversity cluster analysis mostly uses the hierarchical clustering analysis method, which represents the correlation between samples using a hierarchical tree. Figure 2 It can be seen that the Helicobacter flora in the Control group and the T1, T2, and T3 groups are significantly dominant, with absolute richness, while the number in the Model group is significantly reduced and no longer has flora dominance. This indicates that the Model group can reduce the diversity of the dominant flora. The flora in the T1 group is unevenly distributed compared with the T2 and T3 groups, while the flora distribution in the T2 and T3 groups is more uniform. This indicates that the addition of Mishitong Capsules improves the uniformity of the intestinal flora. Hierarchical cluster analysis shows that the addition of Mishitong Capsules can improve the diversity and richness of the dominant flora. And the effect of Mishitong Capsules in improving the uniformity of the intestinal flora is more obvious than that of Shenshitong Granules.

[0046] 2.3.2 PCoA analysis

[0047] like Figure 3 As shown in the figure, PCo1 and PCo2 in the weighted unifrac distance algorithm were 44.7% and 22.7%, respectively. The Model group showed a significant shift in the positive direction of the first principal component, and there was a significant distance between the Model group and the T2 and T3 groups, indicating that the drug-treated group was able to reverse the positive changes caused by kidney stones.

[0048] 2.4 Community column chart analysis

[0049] like Figure 4, as shown in Figure 5, the relative abundance distribution of species composition in each group was plotted at the phylum and genus levels. At the phylum level, the dominant bacterial communities in the five groups were Bacteroidetes, Firmicutes, and Proteobacteria, but the proportions of these three phyla in each group were different. In the Control group, the proportions of the three phyla were 42.63%, 29.87%, and 23.64%, respectively; in the Model group, the proportions were 71.59%, 23.72%, and 2.65%, respectively; in the T1 group, the proportions were 25.47%, 42.63%, and 26.09%, respectively; in the T2 group, the proportions were 45.97%, 32.20%, and 18.32%, respectively; and in the T3 group, the proportions were 54.19%, 35.38%, and 5.02%, respectively. This shows that the three phyla are evenly distributed in the Control, T1, and T2 groups.

[0050] At the genus level, the dominant genus species and proportions in the Control group were Lactobacillus 10.35%, Helicobacter 13.38%, Odoribacter 5.06%, Prevotella 4.26%, and Bacteroides 6.45%, respectively; the dominant genus species and proportions in the Model group were Lactobacillus 18.69%, Odoribacter 4.92%, Prevotella 3.90%, and Bacteroides 6.67%, respectively; the dominant genus species and proportions in the T1 group were Lactobacillus 18.28%, Helicobacter 13.38%, Odoribacter 5.06%, Prevotella 4.26%, and Bacteroides 6.45%, respectively. The dominant genera in group T2 were Lactobacillus 8.18%, Helicobacter 9.22%, Odoribacter 5.68%, Prevotella 5.52%, and Bacteroides 3.05%, respectively; the dominant genera in group T3 were Lactobacillus 8.30%, Helicobacter 1.52%, Odoribacter 5.06%, Prevotella 9.31%, and Bacteroides 5.18%, respectively. The results showed that Mishitong Capsules can effectively reverse the decrease of Prevotella, improve the increase of Bacteroides flora, and increase the new dominant genus Helicobacter.

[0051] 2.5 Species Venn diagram analysis

[0052] The Venn diagram can reflect the number of unique or shared species OTUs in the group, and can also show the overlap of microorganisms between samples and groups, and observe the similarity of the microbiota between groups. Figure 6It can be seen that the number of OTUs in the Control group, Model group, T1, T2, and T3 groups were 1862, 1099, 1407, 1328, and 1736, respectively. The number of OTUs in common among the five groups was 214, the number of OTUs in common between the Model group and the Control group was 181, the number of OTUs in common between the Model group and the T2 and T3 groups was 194 and 182, respectively, and the number of OTUs in common between the Control group and the T2 and T3 groups was 230 and 189, respectively, indicating that the T2 group and the Control group had a high degree of familiarity and similar flora composition, and the number of OTUs in the T2 group was significantly higher than that in the Model group, and the T2 group could increase the diversity of intestinal flora. This shows that the addition of Mishitong capsules can improve the diversity of flora to varying degrees, which is consistent with the analysis results of Beta diversity.

[0053] 2.6 Heatmap Analysis

[0054] The present invention adopts the method of species clustering and makes it into a heat map, and reflects the species richness through color differences. The results show that there are differences in the dominant genera of species richness in the five groups. Figure 7 As shown, the dominant genera of T2, T3 and Model groups were significantly different. The dominant genera of T2 and T3 groups were AF12, Ruminococcus, Mucispinillum, Coprococcus, Rosebura, Dehalobacterum, Odoribacter and other 10 genera, while the dominant genera of Model group were Adlercreutzia, Parabacteroides, Prevotella 3 genera. And the abundance of these dominant genera in Model group decreased significantly. It shows that Model group can reduce the diversity and abundance of dominant genera, while T2 and T3 groups reversed the situation, increasing the diversity of flora and the richness of flora. It shows that the addition of Mishitong capsule can significantly improve the structure of intestinal flora.

[0055] 2.7 PCA analysis

[0056] In the PCA principal component analysis, the present invention analyzes the similarity or difference of the community composition between the intestinal flora samples at the level of mouse flora. Figure 8 The PCA analysis results showed that at the genus level, PC1 and PC2 were 53.2% and 40.4%, respectively. The T2 and T3 groups showed an obvious shift along the negative direction of the first principal component (PC1). There was a relatively obvious distance between the Model group and the T2 and T3 groups.

[0057] The present invention discusses the diversity, richness and uniformity of intestinal flora through Alpha diversity analysis, hierarchical cluster analysis, PCoA analysis, community column chart (phylum and genus level) analysis, species Venn diagram analysis, heat map analysis, PCA analysis, etc. Among them, Bacteroidetes, Firmicutes, and Proteobacteria are dominant phyla. Mishitong Capsules can regulate the intestinal flora of mice by increasing the proportion of Firmicutes and Proteobacteria. The results of various analyses show that the addition of Mishitong Capsules and the increase in dosage can effectively improve the diversity, richness and uniformity of intestinal flora, thereby improving the structure of intestinal flora and maintaining the balance of intestinal flora. On this basis, the metabolism and immunity of the human body are regulated, such as promoting intestinal absorption, inhibiting the occurrence of inflammation, etc. In summary, Mishitong Capsules can be used in the preparation of drugs for regulating the structure of intestinal flora.

[0058] Finally, it should be noted that the above examples are only some specific examples of the invention. Obviously, the invention is not limited to the above embodiments, and there may be many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. The Mishitong capsule is used in the preparation of a drug for treating or regulating intestinal flora disorder, wherein the intestinal flora disorder refers to the intestinal flora disorder caused by kidney stones.

2. The use according to claim 1, characterized in that: The Mishitong capsule is used to improve the diversity, richness and uniformity of intestinal flora.

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