Application of Parvibacter genus in the preparation of products for treating obesity and its related metabolic diseases

Abstract

The present invention discloses the use of Parvibacter in the preparation of products for treating obesity and its related metabolic diseases. The present invention first discloses the use of Parvibacter in the preparation of products for improving obesity-induced insulin resistance or preventing and / or treating obesity and its related metabolic diseases. Further disclosed is a microbial preparation for improving obesity-induced insulin resistance or preventing and / or treating obesity and its related metabolic diseases. The Parvibacter caecicola of the present invention can reduce the blood glucose level of mice with obesity and insulin resistance models induced by a high-fat diet, and can also improve adipose tissue inflammation in mice and reduce fatty degeneration of liver tissue. It plays an important role in the treatment of obesity, type 2 diabetes and non-alcoholic fatty liver disease, and is expected to become a new approach for the treatment of obesity and its related metabolic diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically, to the use of Parvibacter in preparing products for treating obesity and related metabolic diseases. Background Art

[0002] In recent years, the relationship between intestinal microorganisms and obesity and insulin resistance has become a research hotspot. The diversity of intestinal microbial composition in obese patients changes, and the ratio of beneficial bacteria to harmful bacteria is seriously unbalanced, which further promotes the progression of diseases such as obesity and insulin resistance. Currently, the main means of treating obesity include lifestyle intervention, weight loss drugs and metabolic surgery. Lifestyle intervention has limited weight loss, and weight loss drugs have many adverse reactions; although metabolic surgery has good short-term effects in the treatment of obesity, it has certain risks and limitations, and its long-term effectiveness and safety need to be evaluated. Therefore, existing treatment methods are difficult to effectively improve the obesity epidemic.

[0003] The gut microbiome has been a hot research area in recent years. A growing number of studies have shown that imbalances in gut microbiome homeostasis play a key role in the development and progression of obesity and a range of related metabolic diseases, such as non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (T2DM). However, the relationship between diet, gut microbiota, and obesity remains unclear, and specific reports on the relationship between obesity-induced gut microbiome changes and disease progression are lacking. How to specifically modulate gut microbiome composition to improve the progression of insulin resistance, obesity, and related metabolic diseases remains a challenging issue.

[0004] Currently, there are no reports on the therapeutic effects of Parvibacter in improving obesity-induced insulin resistance, obesity and related metabolic diseases. Summary of the Invention

[0005] One object of the present invention is to provide a use of Parvibacter in the preparation of a product for improving obesity-induced insulin resistance and / or treating obesity and its related metabolic diseases.

[0006] Another object of the present invention is to provide a product for improving obesity-induced insulin resistance and / or treating obesity and its related metabolic diseases.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention first provides any one of the following applications of the genus Parvibacter:

[0009] A1) Use in the preparation of a product for improving obesity-induced insulin resistance;

[0010] A2) Use in the preparation of products for preventing and / or treating obesity and related metabolic diseases.

[0011] Furthermore, the bacterium of the genus Parvibacter is Parvibacter caecicola (hereinafter referred to as P.caecicola). Specifically, the Parvibacter caecicola is P.caecicola strain (CCUG57646), purchased from the Swedish CCUG Collection Center.

[0012] Furthermore, the obesity-related metabolic diseases include type 2 diabetes and / or non-alcoholic fatty liver disease.

[0013] Furthermore, the product may be a medicine or a microbial preparation, etc.

[0014] The present invention further discloses a microbial preparation for improving obesity-induced insulin resistance or preventing and / or treating obesity and related metabolic diseases, wherein the active ingredient includes Parvibacter or its fermentation broth or bacterial suspension or culture or its metabolites.

[0015] Furthermore, the bacterium of the genus Parvibacter is Parvibacter caecicola (hereinafter referred to as P.caecicola). Specifically, the Parvibacter caecicola is P.caecicola strain (CCUG57646), purchased from the Swedish CCUG Collection Center.

[0016] Furthermore, the obesity-related metabolic diseases include type 2 diabetes and / or non-alcoholic fatty liver disease.

[0017] In the present invention, the abundance of Parvibacter bacteria was significantly reduced in the intestines of mice with high-fat diet-induced obesity and insulin resistance models. After a hypoglycemic drug was used to lower blood sugar and body weight and improve insulin resistance in the mice, the abundance of Parvibacter bacteria in the intestines of the mice increased significantly. Supplementing P. caecicola suspensions with mice with high-fat diet-induced obesity and insulin resistance models significantly reduced the mice's obesity and insulin resistance, demonstrating that P. caecicola can effectively improve obesity-induced insulin resistance and treat obesity and obesity-related metabolic diseases.

[0018] The beneficial effects of the present invention are as follows:

[0019] The P. caecicola strain of the present invention can reduce blood glucose levels in mice with obesity and insulin resistance induced by a high-fat diet, playing an important role in the treatment of obesity and type 2 diabetes. P. caecicola can also improve adipose tissue inflammation and reduce liver steatosis in mice with obesity and insulin resistance induced by a high-fat diet, playing an important role in the treatment of obesity and non-alcoholic fatty liver disease. Therefore, this invention provides a new theoretical basis for the future use of P. caecicola alone or in combination to improve insulin resistance, obesity, and related metabolic diseases, and is expected to become a new approach for the treatment of obesity and related metabolic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0021] Figure 1 Figure 3 shows the intestinal 16S sequencing results of NCD and HFD mice (A) and the abundance of Parvibacter genus in the intestinal flora of NCD, HFD and HFDA mice (B).

[0022] Figure 2 These are the metabolic index test results of HFD+PARV_C and HFD+PARV mice; where A is body weight, B is the fasting blood glucose level of the mice; C is the plasma glucose concentration 30, 60, 90 and 120 minutes after intraperitoneal injection of insulin at 0.75 IU / kg; D is the alanine aminotransferase (ALT) result in the mouse serum.

[0023] Figure 3 The following are the pathological results of adipose tissue of mice observed by HE staining; among them, A is the pathological result of adipose tissue of NCD mice, B is the pathological result of adipose tissue of HFD mice, C is the pathological result of adipose tissue of HFD+PARVA_C mice, and D is the pathological result of adipose tissue of HFD+PARV mice.

[0024] Figure 4 HE staining was used to observe the liver tissue pathological results of mice (A) and the non-alcoholic fatty liver disease activity score (B). DETAILED DESCRIPTION

[0025] In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments and accompanying drawings. Similar components in the accompanying drawings are represented by the same reference numerals. It should be understood by those skilled in the art that the following detailed description is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0026] In the following examples, the P. caecicola bacteria were P. caecicola strains (CCUG 57646), purchased from the CCUG Collection in Sweden.

[0027] Example 1 Application of P. caecicola in Improving Insulin Resistance and Treating Obesity and Related Metabolic Diseases

[0028] 1. Parvibacter is closely associated with obesity and insulin resistance

[0029] 6-8 week old SPF male wild-type C57BL / 6 mice were selected and housed in a normal environment for 7 days to adapt to the environment.

[0030] in,

[0031] Normal control group (NCD): mice were fed with normal diet for 20 weeks and had free access to water;

[0032] Obesity and insulin resistance model group (HFD): Mice were fed a high-fat diet (60% high-fat diet, purchased from Research Diets, catalog number D12492) for 16 weeks to establish an obesity and insulin resistance model. After successful model establishment (20% increase in body weight compared with the normal control group, and a statistically significant difference in the area under the OGTT curve compared with the normal control group), they were randomly divided into two groups, namely HFDA and HFD. Among them, the mice in the HFDA group had free access to water, and each mouse was given acarbose (ACA) (dose: 500 mg / kg) added to the drinking water daily for 4 weeks; the mice in the HFD group had free access to water for 4 weeks.

[0033] After 20 weeks of feeding, the mice were fecally collected using the stimulated defecation method on the experimental day. If the stimulated defecation method was unsuccessful, the mice were placed in a sterile cage without bedding. Fecal samples were collected immediately after defecation and placed in 1.5 ml EP tubes. 4-5 fecal samples were collected from each mouse. After collection, the mouse fecal specimens were transported in liquid nitrogen as soon as possible and then frozen in a -80°C freezer.

[0034] 16S rDNA sequencing (Illumina HiSeq 2000) was used to analyze the biodiversity of mouse feces, detect the composition of intestinal flora, and observe the differences in the abundance of intestinal flora in each group of mice. Figure 1 As shown. Among them:

[0035] The intestinal 16S sequencing results of NCD and HFD mice are as follows Figure 1 As shown in Figure A, comparison revealed that NCD and HFD mice had significantly different intestinal flora compositions.

[0036] The abundance of Parvibacter genus in the intestinal flora of NCD, HFD and HFDA mice is shown in the following figure: Figure 1As shown in Figure B, the abundance of Parvibacter was significantly decreased in HFD mice compared to NCD mice. However, the abundance of Parvibacter was significantly increased in HFDA mice compared to HFD mice, indicating that acarbose (ACA) treatment can significantly ameliorate the decrease in Parvibacter caused by a high-fat diet.

[0037] These results indicate that a high-fat diet, while causing obesity and insulin resistance in mice, disrupts the balance of their intestinal flora, particularly causing a decrease in the abundance of the genus Parvibacter. However, administration of the type 2 diabetes drug ACA significantly restored the abundance of Parvibacter, suggesting a close association between intestinal Parvibacter and obesity and insulin resistance.

[0038] 2. Construction of a high-fat diet-induced obesity and insulin resistance model group, and oral gavage treatment

[0039] 6-8 week old SPF male wild-type C57BL / 6 mice were selected and housed in a normal environment for 7 days to acclimate. They were randomly divided into four groups: a normal control group (NCD), an obesity and insulin resistance model group (HFD), an HFD + killed bacteria control group (HFD + PARV_C), and an HFD + live bacterial suspension group (HFD + PARV). Body weights were recorded. The mice were housed in an SPF animal facility at a temperature of 20-26°C, a humidity of 40% to 70%, good ventilation, and a 12-hour white-black rotation lighting system.

[0040] in,

[0041] Normal control group (NCD): After mice were fed with normal diet for 16 weeks, each mouse was gavaged with 200 μl of sterile water once a day for 4 weeks.

[0042] Obesity and insulin resistance model group (HFD): Mice were fed a high-fat diet (60% high-fat diet, purchased from Research Diets, Catalog No. D12492) for 16 weeks to establish an obesity and insulin resistance model. After successful model establishment (20% weight gain compared to the normal control group and statistically significant difference in the area under the OGTT curve compared to the normal control group), each mouse was gavaged with 200 μl of sterile water once daily for 4 weeks.

[0043] HFD+PARV_C control group: mice were fed a high-fat diet (60% high-fat diet, purchased from ResearchDiets, catalog number D12492) for 16 weeks to establish an obesity and insulin resistance model. After successful model establishment (weight gain of 20% compared with the normal control group, and the area under the OGTT curve was statistically different from that of the normal control group), each mouse was gavaged with 200 μl of inactivated P. caecicola (Sweden CCUG 57646) suspension (dose: 10 8 CFU / mouse) once a day for 4 weeks.

[0044] HFD+PARV group: mice were fed a high-fat diet (60% high-fat diet, purchased from Research Diets, catalog number D12492) for 16 weeks to establish an obesity and insulin resistance model. After successful model establishment (weight gain of 20% compared with the normal control group, and the area under the OGTT curve was statistically different from that of the normal control group), each mouse was given 200 μl of P. caecicola (Sweden CCUG 57646) bacterial suspension (cultured in liquid culture medium under anaerobic conditions to prepare a bacterial suspension (dose: 10 8 CFU / mouse) by oral gavage once a day for 4 weeks.

[0045] 3. Detection and Analysis of Metabolic and Pathological Indicators

[0046] 1. Detection of metabolic indicators in mice

[0047] The body weight of mice in each group was measured once a week. The body weight and average values of the HFD+PARV_C group and HFD+PARV group after 4 weeks of oral administration of bacterial suspension were as follows: Figure 2 As shown in middle A, the results showed that compared with HFD+PARVA_C mice, HFD+PARV mice had lower body weight, indicating that P. caecicola can significantly reduce the weight gain of mice caused by high-fat diet.

[0048] Fasting blood glucose was measured once a week. After successful model establishment and 4 weeks after bacterial suspension supplementation, mice in each group underwent IPITT (intraperitoneal injection of insulin 0.75u / kg, purchased from NovoNordisk, catalog number 202106ABF1) test. Blood samples were collected by tail clipping before and 30, 60, 90 and 120 minutes after insulin injection, and blood glucose concentration was measured. The results are shown in the table below. Figure 2 As shown in B and C, the results showed that compared with HFD+PARV_C mice, the fasting blood glucose of HFD+PARV mice was significantly decreased ( Figure 2 Middle B), and increased insulin sensitivity ( Figure 2Middle C), indicating that P. caecicola can significantly improve the increased blood sugar and insulin resistance of mice caused by a high-fat diet.

[0049] Before the mice were killed, they were anesthetized with ether and their eyeballs were removed to collect blood. Serum was extracted and liver function indexes were measured by ELISA. The results showed that compared with HFD+PARV_C mice, the alanine aminotransferase (ALT) of HFD+PARV mice was significantly decreased ( Figure 2 Middle D).

[0050] The above results show that in the high-fat diet-induced obesity and insulin resistance model, which causes obesity and type 2 diabetes in mice, manifested as weight gain, increased blood sugar and decreased insulin sensitivity, after supplementation with P. caecicola suspension, the mice's weight and fasting blood sugar decreased, insulin sensitivity increased, and ALT decreased, indicating that P. caecicola can improve obesity and obesity-induced insulin resistance, lower blood sugar levels, and play an important role in the treatment of obesity and type 2 diabetes.

[0051] 2. Pathological changes in adipose tissue

[0052] Mice were killed by overdose anesthesia or cervical dislocation, and the peri-epididymal adipose tissue was obtained, weighed, and made into paraffin sections. The pathological changes of adipose tissue were observed by HE staining. The adipose tissue pathological results of NCD, HFD, HFD+PARV_C, and HFD+PARV mice were shown in the figure. Figure 3 As shown in Figures A, B, C, and D, the results showed that the adipose tissue of NCD mice was uniform in size and had no inflammatory cell infiltration; compared with NCD mice, the adipose vacuoles of HFD mice were significantly enlarged, accompanied by a large number of inflammatory cell infiltration; compared with HFD mice, the adipose tissue of HFD+PARV_C mice did not show a significant reduction, accompanied by inflammatory cell infiltration; compared with HFD+PARV_C mice, the adipose tissue vacuoles of HFD+PARV mice were significantly smaller, and the inflammatory cell infiltration was reduced.

[0053] These results demonstrate that in a high-fat diet-induced model of obesity and insulin resistance, adipose tissue vacuoles significantly enlarge, accompanied by a large infiltration of inflammatory cells. Administration of a P. caecicola suspension significantly reduced adipose tissue vacuoles and inflammatory cell infiltration, demonstrating that P. caecicola can significantly improve adipose tissue vacuoles and inflammatory infiltration.

[0054] 3. Pathological changes in liver tissue

[0055] Under deep anesthesia, the mouse chest and abdominal cavity were exposed. 30 ml of normal saline was drawn into the syringe to perfuse the liver in vivo until the liver turned white. The gallbladder was cut off and the liver was removed. Part of the liver tissue was made into paraffin sections and the pathological changes of the liver tissue were observed by HE staining. The results are as follows Figure 4 As shown in Figure A, observations at 10x and 20x magnification revealed that compared with NCD mice, HFD mice had a large number of fat vacuoles in the liver tissue, obvious lobular inflammatory infiltration, and obvious ballooning of the liver tissue. Compared with HFD+PARV_C mice, HFD+PARV mice had significantly reduced fat vacuoles in the liver tissue, and significantly improved inflammatory necrotic foci. The results of the non-alcoholic fatty liver disease activity score (NAS) were as follows: Figure 4 As shown in Figure 3B, the results showed that the NAS scores of HFD+PARV mice were significantly decreased compared with those of HFD+PARV_C mice.

[0056] These results demonstrate that a high-fat diet-induced obesity and insulin resistance model induces non-alcoholic fatty liver disease in mice, characterized by the presence of numerous fatty vacuoles and ballooning in liver tissue, along with significant lobular inflammatory infiltration and an elevated Non-alcoholic Fatty Liver Disease Activity Score (NAS) score. Administration of a P. caecicola suspension significantly reduced fatty vacuoles in liver tissue, significantly improved inflammatory necrotic foci, and decreased NAS scores, suggesting that P. caecicola plays an important role in the treatment of obesity and non-alcoholic fatty liver disease.

[0057] In summary, P. caecicola can lower blood sugar levels in mice, thereby playing an important role in the treatment of obesity and type 2 diabetes; P. caecicola can also improve adipose tissue inflammation in mice and reduce fatty degeneration of liver tissue, thereby playing an important role in the treatment of non-alcoholic fatty liver disease.

[0058] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. Strains P. caecicola Use of CCUG 57646 in the preparation of a product for improving obesity-induced insulin resistance.

2. Strains P. caecicola Use of CCUG 57646 in the preparation of a product for treating obesity, type 2 diabetes and / or non-alcoholic fatty liver disease.

3. A microbial preparation, characterized in that The active ingredients of the microbial preparation include strains P. caecicola CCUG 57646 or its fermentation broth or bacterial suspension.

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