Application of parabacteroides dielsii in preparation of medicine for improving CKD sarcopenia

Abstract

The invention discloses application of parabacteroides dieldrii in preparation of a medicine for improving CKD sarcopenia, and belongs to the technical field of microorganisms. According to the application, blood-targeted metabonomics, fecal metagenomics, muscle-targeted metabonomics and muscle transcriptomics detection and analysis are carried out on a constructed CKD model mouse, and it is found that bile acid and lipid metabolism abnormalities exist in muscle tissues of the CKD sarcopenia mouse; in combination with metagenomics, it is further found that the parabacteroides dieldrii in the intestinal flora of the mouse is closely associated with metabolism of the parabacteroides dieldrii and the intestinal flora of the mouse. After the parabacteroides dieldrii is supplemented into the CKD model mouse body, the expression levels of bile acid and lipid metabolism related receptors can be intervened, so that the metabolism of the bile acid and lipid metabolism related receptors is improved, and the muscle quality of the mouse is obviously improved. In conclusion, the invention provides the new application of the parabacteroides dieldrii in preparing the medicine for improving the CKD sarcopenia.

Description

Technical Field

[0001] The present application belongs to the field of microbial technology, and specifically relates to the use of Parabacteroides distichum in the preparation of drugs for improving CKD sarcopenia. Background Art

[0002] Chronic kidney disease (CKD) is a multifactorial disorder of renal structure and function. Its complications affect multiple systems and organs, severely impacting patients' quality of life and life expectancy. Sarcopenia is a systemic, progressive, age-related skeletal muscle disorder characterized by loss of skeletal muscle mass and function. It is a common complication in CKD patients. Reduced muscle protein synthesis and increased muscle protein breakdown lead to excessive skeletal muscle loss, predisposing patients to sarcopenia. Compared with age-matched non-CKD patients, the prevalence of sarcopenia in CKD patients is significantly higher, accounting for approximately 4% to 42% of CKD patients. The specific incidence is correlated with the degree of renal function deterioration and CKD stage. CKD combined with sarcopenia not only increases the risk of progression to end-stage renal disease but also the incidence of cardiovascular disease and all-cause mortality. Furthermore, sarcopenia increases the risk of disability from diseases such as falls and fractures, as well as hospitalization, significantly reducing the quality of life and increasing the economic burden of CKD patients. However, there is currently no effective treatment for sarcopenia in CKD.

[0003] Intervention measures for patients with CKD sarcopenia were once mainly based on nutritional therapy and exercise. However, high-protein intake may increase the burden on the kidneys of CKD patients, while the nutritional supply of branched-chain amino acids and ketoacid preparations is limited. At the same time, CKD patients often suffer from symptoms such as fatigue and weakness, making it difficult for them to persist in high-intensity exercise. With the rapid development of the field of intestinal microorganisms, the theory that intestinal microbial imbalance can affect muscle metabolism and function has been proposed. Therefore, probiotics, prebiotics or synbiotics are now used to regulate the balance of intestinal flora and thus improve CKD sarcopenia. However, because the specific mechanism of the relationship between intestinal microorganisms and sarcopenia is still unclear, there is still a lack of scientific basis for the selection of probiotic strains and dosage control. As a result, the treatment of CKD sarcopenia is not targeted enough, and the therapeutic effect of using probiotics, prebiotics or synbiotics is still limited.

[0004] Therefore, exploring the correlation between intestinal microorganisms and CKD sarcopenia and accurately regulating the balance of intestinal microorganisms are of great significance for achieving targeted treatment of CKD sarcopenia. Summary of the Invention

[0005] 1. Problem to be solved

[0006] The correlation between CKD sarcopenia and intestinal microorganisms is still unclear. The current use of probiotics, prebiotics or synbiotics to regulate intestinal flora has problems such as lack of pertinence and limited therapeutic effect. This application constructs CKD model mice and analyzes their metabolomics. It is found that there are abnormalities in bile acid and lipid metabolism in the muscle tissue of sarcopenic mice. Combined with metagenomics, it is further revealed that the metabolism of the two is closely related to the mouse intestinal flora, Parabacteroides dieldrinii. This application supplements CKD model mice with Parabacteroides dieldrinii and finds that the muscle quality of the mice is significantly improved. It then proposes the use of Parabacteroides dieldrinii for the preparation of new medicines to improve CKD sarcopenia.

[0007] 2. Technical solution

[0008] This application provides the use of Parabacteroides dieldrinii in the preparation of a drug for improving CKD sarcopenia. This application analyzes the muscle tissue targeted metabolomics of CKD model mice and finds abnormalities in bile acid and lipid metabolism in the muscle tissue of CKD sarcopenia mice. Combined with the analysis of fecal metagenomics and blood targeted metabolomics detection results of CKD model mice, it is found that Parabacteroides dieldrinii has a strong correlation with bile acid and lipid metabolism. By supplementing Parabacteroides dieldrinii with CKD model mice, the muscle quality of the mice was significantly improved, thus proposing a new medical use of Parabacteroides dieldrinii for improving CKD sarcopenia.

[0009] Furthermore, the above-mentioned Parabacteroides distasonis has a deposit number of ATCC 8503, which is recorded in the prior art Zhao Q, Dai MY, Huang RY, et al. Parabacteroides distasonis ameliorates hepaticfibrosis potentially via modulating intestinal bile acid metabolism and hepatocyte pyroptosis in male mice. Nat Commun. 2023; 14(1): 1829. Published 2023Apr 1. doi: 10.1038 / s41467-023-37459-z, but in the application document, its function is to improve liver fibrosis by regulating bile acid metabolism and hepatocyte pyroptosis.

[0010] The present application also provides a drug for improving CKD sarcopenia, which comprises the above-mentioned Parabacteroides dieldrinii.

[0011] Furthermore, the deposit number of the Parabacteroides distichum used above is ATCC 8503.

[0012] Furthermore, the above-mentioned medicine also includes pharmaceutically acceptable excipients.

[0013] Furthermore, the dosage form of the above-mentioned pharmaceutical preparation includes any one of a liquid preparation, a solid preparation, and a semisolid preparation.

[0014] 3. Beneficial effects

[0015] Compared with the prior art, the present application has the following advantages:

[0016] (1) The application of Parabacteroides dieldrinii provided in this application in the preparation of drugs for improving CKD sarcopenia. By constructing CKD model mice, the detection and analysis of blood-targeted metabolomics, fecal metagenomics, muscle-targeted metabolomics and muscle transcriptomics were completed. It was found that there were problems of abnormal bile acid and lipid metabolism in the muscle tissue of CKD sarcopenia mice. Combined with metagenomics, it was further noted that Parabacteroides dieldrinii in the mouse intestinal flora had a strong correlation with the metabolism of the two. By supplementing Parabacteroides dieldrinii into the CKD model mice, it was found that the muscle mass of the mice was significantly improved, confirming the effectiveness of the intestinal flora Parabacteroides dieldrinii for the treatment of skeletal muscle atrophy. For the first time, it was proposed that Parabacteroides dieldrinii be used to prepare a new medicine for improving CKD sarcopenia.

[0017] (2) The application of Parabacteroides dieldii provided in this application for the preparation of a drug for improving CKD sarcopenia. The Parabacteroides dieldii used is a natural symbiotic bacterium in the intestine. As one of the core flora of the human body, it has unique metabolic and immune regulatory functions. When used to improve skeletal muscle atrophy in patients with CKD sarcopenia, on the one hand, it will not cause kidney burden in CKD patients and is highly safe; on the other hand, compared with existing probiotics, prebiotics or synbiotics, the use of Parabacteroides dieldii to regulate intestinal microbial balance for improving muscle quality is more targeted.

[0018] (3) The application of Parabacteroides dissimilars provided in this application in the preparation of drugs for improving CKD sarcopenia, by immunoblotting experiments (Western Blot) to detect the expression levels of bile acid receptors TGR5, FXR and lipid metabolism-related transcription factors PPARα, PPARγ in the muscle tissue of CKD model mice before and after supplementation with Parabacteroides dissimilars, respectively, and found that Parabacteroides dissimilars intervened in the expression levels of the above receptors, thereby achieving improvements in bile acid and lipid metabolism. This application further reveals the mechanism of action of Parabacteroides dissimilars in improving CKD sarcopenia at the molecular level, and also lays an important foundation for the research and development of more CKD sarcopenia drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1Comparison of renal function, body weight, grip strength, and muscle between sham-operated mice (Sham) and CKD mice: (A) serum creatinine; (B) blood urea nitrogen; (C) body weight; (D) grip strength; (E) skeletal muscle wet weight; (F) muscle comparison; (G) tibialis anterior muscle CSA; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0020] Figure 2 This is a comparative diagram of fecal metagenomic analysis between sham-operated mice (Sham) and CKD mice: (A) Chao1 index; (B) Shannon index; (C) PCA analysis; (D) species composition analysis - phylum level; (E) species composition analysis - genus level; (F) species composition analysis - species level; (G) relative abundance of Bacteroidetes; (H) Firmicutes / Bacteroidetes; (I) relative abundance of Parabacteroides genus; (J) relative abundance of Parabacteroides distichum; *P<0.05, **P<0.01.

[0021] Figure 3 Figure 3. Flowchart of the experimental study on the supplementation of Parabacteroides distichum in sham-operated mice (Sham) and CKD mice with Parabacteroides distichum, and the comparison of body weight and muscle mass of mice after supplementation: (A) Experimental process; (B) Muscle comparison; (C) Grip strength; (D) Body weight; (E) Skeletal muscle wet weight; (F) Skeletal muscle dry weight; (G) CSA; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0022] Figure 4 Comparison of blood metabolomics analysis between sham-operated mice (Sham) and CKD mice: (A) PCA analysis; (B) OPLS-DA analysis; (C) volcano plot of differential metabolites; (D) primary bile acid levels; (E) secondary bile acid levels; (F) heat map of correlation analysis between intestinal flora and bile acids; *P<0.05, **P<0.01, ***P<0.001.

[0023] Figure 5 Comparison of bile acid and receptor expression levels in muscle tissue between sham-operated mice and CKD mice: (A) Primary bile acid levels; (B) Secondary bile acid levels; (C) Western blot analysis of TGR5 expression in muscle tissue; (D) Western blot analysis of FXR expression in muscle tissue. *P < 0.05, **P < 0.01.

[0024] Figure 6Comparison of metabolomics and transcriptomics analysis of muscle tissues of sham-operated mice (Sham) and CKD mice: (A) PCA analysis of the metabolome; (B) OPLS-DA analysis of the metabolome; (C) volcano plot of differential metabolites; (D) KEGG enrichment bubble plot of the metabolome; (E) OPLS-DA analysis of the transcriptome; (F) volcano plot of differential genes; (G) KEGG enrichment bubble plot of the transcriptome.

[0025] Figure 7 Comparison of lipid metabolism pathways in muscle tissue between sham-operated mice and CKD mice: (A) Muscle BODIPY staining; (B) Western blot analysis of PPARα protein expression in muscle tissue; (C) Western blot analysis of PPARγ protein expression in muscle tissue. *P < 0.05.

[0026] Figure 8 This is a comparison of the changes in bile acid receptors in mouse muscle tissue after supplementation with Parabacteroides dieldrinii: (A) WB detection of FXR expression level in muscle tissue; (B) WB detection of TGR5 expression level in muscle tissue; *P<0.05.

[0027] Figure 9 This is a comparison chart of the detection of lipid metabolism-related pathways in mouse muscle tissue after supplementation with Parabacteroides dieldrinii: (A) muscle BODIPY staining; (B) WB detection of PPARα protein expression level in muscle tissue; (C) WB detection of PPARγ protein expression level in muscle tissue; *P<0.05. DETAILED DESCRIPTION

[0028] The present application is further described below with reference to specific embodiments.

[0029] Example 1

[0030] This example provides the construction of CKD model mice, and the specific procedures are as follows:

[0031] (1) Surgical treatment:

[0032] Eight-week-old healthy male C57BL / 6 mice with the same growth conditions were adaptively raised under the same conditions for one week and then randomly divided into two groups for surgical treatment. One group served as the control group, i.e., the sham operation group, and the other group served as the experimental group and underwent 5 / 6 nephrectomy to induce renal function damage and obtain CKD model mice.

[0033] The specific nephrectomy procedure is as follows:

[0034] The first operation was performed under a strict disinfection environment. After anesthesia, the mouse was fixed in a prone position. A longitudinal incision of about 1 cm was made in the renal anatomical area on the left side of the back, and about 2 / 3 of the upper and lower poles of the kidney were removed. After hemostasis and suture, the first operation was completed. One week later, when the mouse had fully recovered, the second operation was performed. A longitudinal incision was made in the renal anatomical area on the right side of the back, the surrounding fat tissue was freed and ligated with silk thread at the renal hilum, and then the entire right kidney was removed.

[0035] (2) Index detection:

[0036] Eight weeks after surgery, the sham-operated mice and CKD model mice were evaluated for renal function impairment, body weight, and muscle mass. Specifically:

[0037] Renal function impairment detection: Mouse blood creatinine and blood urea nitrogen were tested using the creatinine determination kit (C011-2-1) and urea nitrogen test kit (C013-2-1) from Nanjing Jiancheng Bioengineering Institute to assess the degree of renal function impairment. Specific procedures were performed according to the kit instructions.

[0038] Body weight: Use a high-precision electronic scale to weigh the mice.

[0039] Grip strength: A gripper produced by Nanjing Calvin was used to fix the mouse's limbs to a grid. The mouse's tail was dragged until the limbs completely left the grid. The sensor waveform changes recorded the changes in the mouse's grip strength during the process. The peak value was used as the mouse's single grip strength. Each mouse was measured five times, and the average of the three highest values was taken as the mouse's final grip strength value.

[0040] Muscle mass: The wet weights of the gastrocnemius, tibialis anterior, soleus, plantar, and extensor digitorum longus (EDL) muscles were measured using an analytical balance.

[0041] (3) Result analysis:

[0042] The results are as follows Figure 1 As shown in Figure 2, compared with the sham operation group, the blood creatinine and blood urea nitrogen levels of the mice in the experimental group were significantly increased ( Figure 1 A, B), significant weight loss ( Figure 1 Middle C), grip strength decreased ( Figure 1 Middle D), muscle mass decreased ( Figure 1 E, F), the wet weights of gastrocnemius, tibialis anterior and soleus muscles showed statistically significant differences compared with the sham operation group ( Figure 1Middle E); Laminin staining was performed on frozen sections of the tibialis anterior muscle. The results showed that the average cross-sectional area (CSA) of the muscle fibers in the experimental group was lower than that in the sham-operated group. Compared with the sham-operated group, the frequency distribution of the cross-sectional area of the muscle fibers in the experimental group was significantly shifted to the left (the cross-sectional area became smaller) ( Figure 1 Middle G).

[0043] In summary, by comparing various indicators with those of the sham-operated group mice, this example successfully obtained CKD sarcopenia mice through 5 / 6 nephrectomy surgery.

[0044] Example 2

[0045] This example provides an analysis of fecal metagenomics results from sham-operated mice and CKD model mice.

[0046] Fresh fecal samples from the sham-operated mice and CKD model mice were collected 8 weeks after surgery and stored at -80°C for future use.

[0047] The fecal metagenomics test in this example was completed by Wuhan Maiwei Metabolic Biotechnology Co., Ltd., and the test results are analyzed as follows:

[0048] The Chao1 index and Shannon index of the CKD group mice were significantly higher than those of the sham-operated group mice, indicating that there were significant differences in the α diversity, i.e., species richness and evenness, between the two groups of mice ( Figure 2 Principal component analysis (PCA), representing β diversity, showed that the composition and abundance of the microbial communities in the CKD group and the sham-operated group were significantly different ( Figure 2 C). Species composition analysis showed that the intestinal flora of the two groups of mice were different at the phylum level ( Figure 2 D), genus level ( Figure 2 E) and species level ( Figure 2 There were differences in the intestinal flora of CKD model mice.

[0049] Specifically, the results at the phylum level showed that Bacteroidota, Firmicutes, and Pseudomonadota were the dominant phyla. Among them, the relative abundance of Bacteroidetes in the CKD group was significantly lower than that in the sham operation group ( Figure 2 The relative abundance of Firmicutes in the CKD group was higher than that in the sham group, but there was no statistical difference. The ratio of Firmicutes to Bacteroidetes is a sign of intestinal flora imbalance. The ratio of Firmicutes to Bacteroidetes in the CKD group showed a significant increase ( Figure 2 Middle H).

[0050] Specifically, the genus-level results showed that Bacteroides, Prevotella, Alistipes, and Parabacteroides were the dominant genera. Among them, the relative abundances of Bacteroides, Prevotella, and Alistipes were not statistically different between the two groups of mice. Only the relative abundance of Parabacteroides was different between the two groups, and it was significantly decreased in the CKD group ( Figure 2 Among the genus Parabacteroides, the relative abundance of Parabacteroides distichous decreased most significantly in the CKD group ( Figure 2 (J).

[0051] In summary, through the fecal metagenomic analysis of sham-operated mice and CKD model mice, at the genus level, attention was paid to Parabacteroides distichous, whose relative abundance was significantly decreased in the intestine of CKD mice.

[0052] Example 3

[0053] This example provides the use of Parabacteroides distichum in improving CKD sarcopenia. It is based on the supplementary experiment of Parabacteroides distichum on sham-operated mice and CKD model mice in Example 2. The specific operation is as follows:

[0054] (1) Oral gavage treatment:

[0055] A total of 4 treatment groups were set up, with 8 mice in each group. The specific gavage treatment of mice in each group was as follows:

[0056] a. Sham operation group (sterile PBS gavage 200 μL / d);

[0057] b. Sham operation + Parabacteroides distichum (Pd) group (concentration of 2×10 8 CFU / ml of Parabacteroides distichum solution 200 μL / d);

[0058] c. CKD group (sterile PBS gavage 200 μL / d)

[0059] d.CKD+ Parabacteroides distichum (Pd) group (concentration of 2×10 8 CFU / ml of Parabacteroides distichum solution 200 μL / d).

[0060] The mice were treated by gavage every day as described above, and were killed after 4 consecutive weeks of treatment. Figure 3 In this example, Parabacteroides distichous was purchased from Ningbo Mingzhou Biotechnology Co., Ltd. with the product number B81085 and the deposit number ATCC 8503.

[0061] (2) Index detection:

[0062] The muscle mass, grip strength, body weight, skeletal muscle wet weight and dry weight (the plantaris muscle, soleus muscle and extensor digitorum longus muscle were treated in a 60°C oven overnight and weighed using an analytical balance to measure the dry weight) of the four groups of mice were tested respectively. The skeletal muscle wet weight test type was the same as in Example 1, and the detection methods of each indicator were the same as in Example 1.

[0063] (3) Result analysis:

[0064] The results are as follows Figure 3 As shown, the muscle mass of CKD model mice increased after intervention with Parabacteroides distichum ( Figure 3 Middle B), improved grip strength ( Figure 3 Middle C), weight gain ( Figure 3 D). Among them, the wet weight of gastrocnemius, tibialis anterior, plantaris and extensor digitorum longus ( Figure 3 E) and the dry weight of soleus and extensor digitorum longus increased significantly compared with those of CKD mice without intervention ( Figure 3 Middle F), muscle fiber CSA also increased significantly compared with the non-intervention group ( Figure 3 Middle G).

[0065] In summary, the experimental results of this example show that Parabacteroides distichous can effectively improve sarcopenia in CKD mice.

[0066] Example 4

[0067] This example provides an analysis of the results of targeted metabolomics testing of the blood of sham-operated mice and CKD model mice.

[0068] Eight weeks after surgery, blood samples were collected from the sham-operated mice and CKD model mice by eye bleeding, and the serum was extracted and stored at -80°C for later use.

[0069] The blood targeted metabolomics test in this example was completed by Wuhan Maiwei Metabolic Biotechnology Co., Ltd., and the test results are analyzed as follows:

[0070] There were significant differences in the metabolite levels between the CKD group and the sham-operated group ( Figure 4 Compared with the sham group, 552 metabolites were significantly increased and 142 metabolites were significantly decreased in the CKD group ( Figure 4 Among them, the levels of primary bile acids (CA, CDCA, UDCA, α-MCA, β-MCA) in mice in the CKD group were significantly increased ( Figure 4 Middle D), secondary bile acid levels (ILCA, 3-oxo-DCA, LCA, 23-DCA) were significantly decreased ( Figure 4E). Species-level correlation analysis between intestinal flora and bile acids showed that Parabacteroides dieffenbachia was significantly negatively correlated with primary bile acids and significantly positively correlated with secondary bile acids, and there was a strong correlation between secondary bile acid LCA and Parabacteroides dieffenbachia ( Figure 4 Middle F).

[0071] In summary, through the targeted metabolomics analysis of the blood of sham-operated mice and CKD model mice, it was found that Parabacteroides distichous was correlated with primary bile acids and secondary bile acids, especially secondary bile acid LCA.

[0072] Example 5

[0073] This example is based on Example 4 and detects the expression levels of bile acids and their receptors in muscle tissues of sham-operated mice and CKD model mice.

[0074] Specifically, this example used muscle-targeted metabolomics to detect bile acid levels in the muscle tissue of CKD mice. Gastrocnemius muscle tissue from sham-operated mice and CKD model mice 8 weeks after surgery was obtained and sent to Wuhan Maiwei Metabolic Biotechnology Co., Ltd. for testing. Western blot was also used to detect the expression levels of bile acid receptors TGR5 and FXR in the muscle tissue of CKD mice. The specific experimental procedures were as follows:

[0075] Gastrocnemius muscle tissue was lysed in RIPA buffer, ground, and sonicated, then centrifuged at 12,000 rpm for 10 minutes. The supernatant was transferred to an EP tube. Protein quantification was performed using the BCA assay. Proteins were denatured in a 70°C waterbath with loading buffer for 10 minutes, cooled to room temperature, and stored at -80°C. Samples were loaded onto a 4-20% SDS-PAGE gel and subjected to electrophoresis at 120 V for 60 minutes. The gel was then transferred to a 0.45 μm PVDF membrane and blocked with blocking buffer (5% skim milk) for 1 hour at room temperature. FXR and TGR5 were then detected using rabbit monoclonal antibodies against NR1H4 (1:1000, Proteintech) and GPBAR1 (1:1000, Proteintech). A mouse monoclonal anti-GAPDH antibody (1:5000, CST) was used as a loading control. After overnight incubation, secondary antibodies (1:2000, Servicebio) were used. Finally, immunoreactive proteins were detected using Western ECL Substrate and quantified using Image J software.

[0076] The results are analyzed as follows:

[0077] Compared with the sham operation group, the levels of primary bile acids (CA, α-MCA, and β-MCA) in the muscle tissue of mice in the CKD group were increased, but there was no statistical difference ( Figure 5Middle A); secondary bile acid (ILCA, isoCDCA, LCA, 6-ketoLCA) levels decreased significantly ( Figure 5 Correspondingly, Western Blot confirmed that the primary bile acid receptor TGR5 ( Figure 5 C) and FXR( Figure 5 Middle (D) expression was downregulated, and the downregulation of FXR was more significant.

[0078] In summary, by detecting the expression levels of bile acids and their receptors in the muscle tissue of sham-operated mice and CKD model mice, it was found that the expression of primary bile acid receptors in muscle tissue was downregulated, and the conversion of primary bile acids to secondary bile acids in the muscle tissue of CKD mice was affected.

[0079] Example 6

[0080] This example provides analysis of muscle-targeted metabolomics and muscle transcriptomics results in sham-operated mice and CKD mice.

[0081] The gastrocnemius muscle tissues of the sham-operated mice and CKD model mice were collected 8 weeks after surgery and stored at -80°C for future use.

[0082] The muscle-targeted metabolomics and muscle transcriptomics tests in this example were performed by Wuhan Maiwei Metabolic Biotechnology Co., Ltd. The test results are analyzed as follows:

[0083] Muscle-targeted metabolomics analysis showed that there were significant differences in muscle metabolite levels between CKD mice and sham-operated mice ( Figure 6 Compared with the sham group, 116 metabolites were significantly increased and 190 metabolites were significantly decreased in the CKD group ( Figure 6 Middle C). The KEGG enrichment pathway bubble diagram showed that lipid metabolism-related pathways glycerophospholipid metabolism and glycerolipid metabolism were significantly enriched in the CKD group ( Figure 6 Middle D).

[0084] Muscle transcriptomic analysis showed that there were significant differences in transcriptomic levels between the CKD group and the sham group ( Figure 6 Middle E), compared with the sham operation group, 26 genes were up-regulated and 179 genes were down-regulated in the CKD group, among which NR1H4, the nuclear receptor gene encoding FXR, was down-regulated ( Figure 6 Middle F). The KEGG enrichment pathway bubble diagram showed that the biosynthesis of fatty acids related to lipid metabolism was significantly changed in the CKD group ( Figure 6 Middle G).

[0085] In summary, through the analysis of muscle-targeted metabolomics and muscle transcriptomics in sham-operated mice and CKD model mice, it was found that the downregulation of FXR was due to the downregulation of the nuclear receptor gene NR1H4 encoding FXR; in addition, there were significant changes in fatty acid biosynthesis in CKD model mice, which led to abnormalities in lipid metabolism-related pathways in the muscle tissue of CKD mice.

[0086] Example 7

[0087] This example is based on Example 6 and detects the expression levels of lipids and transcription factors that regulate their metabolism in muscle tissues of sham-operated mice and CKD model mice.

[0088] Specifically, this example further detected the expression levels of lipids and transcription factors that regulate their metabolism in the muscle tissue of CKD mice by performing BODIPY staining and Western Blot experiments on frozen sections of mouse muscle tissue.

[0089] BODIPY staining was performed as follows: fresh tibialis anterior muscles were isolated and fixed in muscle fixative, and then embedded in OCT. 8 μm thick sections were prepared and stained with BODIPY dye (3.8 μM working solution, Invitrogen).

[0090] The Western blot assay was performed as follows: Prepared gastrocnemius muscle protein samples were loaded onto a 4-20% SDS-PAGE gel and subjected to electrophoresis at 120 V for 60 minutes. The samples were then transferred to a 0.45 μm PVDF membrane and blocked with blocking buffer (5% skim milk) for 1 hour at room temperature. Probing was then performed using rabbit monoclonal antibodies against PPARα (1:1000, Abcam) and rabbit monoclonal antibodies against PPARγ (1:1000, CST). A mouse monoclonal anti-GAPDH antibody (1:5000, CST) was used as a loading control. After overnight incubation, a secondary antibody (1:2000, Servicebio) was used. Immunoreactive proteins were detected using Western ECL substrate and quantified using Image J software.

[0091] The results are analyzed as follows:

[0092] BODIPY staining of frozen sections of muscle tissue showed that the accumulation of neutral lipid droplets in skeletal muscle of mice in the CKD group was more obvious than that in the sham operation group ( Figure 7 Correspondingly, Western Blot confirmed that the expression of PPARα, a key transcription factor regulating lipid metabolism, was downregulated in the CKD group ( Figure 7 Middle B), indicating that fatty acid decomposition decreased; PPARγ expression was upregulated in the CKD group ( Figure 7 Middle C), indicating increased fat storage.

[0093] In summary, compared with the sham-operated mice, the expression of PPARα, a key transcription factor involved in lipid metabolism, was downregulated and PPARγ was upregulated in the muscle tissue of the CKD mice, resulting in decreased fatty acid decomposition and fat accumulation. Therefore, neutral lipid droplet aggregation was observed in the muscle tissue sections, and obvious lipid deposition occurred.

[0094] Example 8

[0095] This example detected the expression levels of bile acid receptors TGR5 and FXR in muscle tissue of the four groups of mice supplemented with Parabacteroides distichum in Example 2, as follows:

[0096] The immunoblotting experiment was the same as in Example 5.

[0097] Result analysis:

[0098] like Figure 8 As shown in the results, after supplementation with Parabacteroides dieldrinii, the expression levels of bile acid receptors FXR and TGR5 in the muscle tissue of CKD model mice were significantly upregulated, indicating that Parabacteroides dieldrinii has an improving effect on the abnormal expression of bile acid receptors FXR and TGR5 in the muscle tissue of mice, thereby promoting the normal metabolism of bile acids.

[0099] Example 9

[0100] This example detected the expression levels of lipids and transcription factors that regulate their metabolism in the muscle tissues of the four groups of mice supplemented with Parabacteroides distichous in Example 2, as follows:

[0101] BODIPY staining and Western Blot experiments were performed on frozen sections of mouse muscle tissue, using the same procedures as in Example 7.

[0102] Result analysis:

[0103] like Figure 9 As shown in the results, after supplementation with Parabacteroides distichous, the accumulation of neutral lipid droplets in the muscle tissue of mice was significantly reduced compared with the non-supplemented group, and this was particularly evident in CKD model mice ( Figure 9 Western Blot confirmed that PPARα expression was upregulated in CKD model mice after intervention with Parabacteroides distichum ( Figure 9 Middle B), indicating increased fatty acid decomposition; PPARγ expression was downregulated ( Figure 9 Middle C), indicating a decrease in fat storage.

[0104] In summary, Parabacteroides distichum improves lipid metabolism in muscle tissue of CKD model mice by intervening in the expression of PPARα and PPARγ.

Claims

1. Application of Parabacteroides distichum in the preparation of drugs for improving CKD sarcopenia.

2. The use of Parabacteroides distichum according to claim 1 in the preparation of a medicament for improving CKD sarcopenia, characterized in that The deposit number of the Parabacteroides distichum is ATCC 8503.

3. A drug for improving CKD sarcopenia, characterized in that: The drug includes Parabacteroides distichum.

4. The drug for improving CKD sarcopenia according to claim 3, characterized in that: The deposit number of the Parabacteroides distichum is ATCC 8503.

5. The drug for improving CKD sarcopenia according to claim 3, characterized in that: The drug also includes pharmaceutically acceptable excipients.

6. The drug for improving sarcopenia in chronic kidney disease according to claim 3, characterized in that: The dosage form of the pharmaceutical preparation includes any one of a liquid preparation, a solid preparation, and a semisolid preparation.

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