阅读说明：本技术 一种改善NAFLD组织病变的地衣芽孢杆菌BL3 Sir2样蛋白及其应用 (Bacillus licheniformis BL3Sir 2-like protein for improving NAFLD (non-alcoholic fatty acid) tissue lesion and application thereof ) 是由 孙晗笑 徐臻睿 利时雨 于 2018-06-19 设计创作，主要内容包括：一种改善NAFLD组织病变的地衣芽孢杆菌BL3 Sir2样蛋白及其应用。本发明公开了一种具有显著改善非酒精性脂肪性肝病组织病变并抑制脂滴形成的地衣芽孢杆菌BL3 Sir2样蛋白及其应用,属于生物技术领域。本发明具有显著改善NAFLD组织病变并抑制脂滴形成的地衣芽孢杆菌BL3 Sir2样蛋白是从<I>Baclicuslicheniformis</I> BL3菌株中分离制备而得,所述菌株由本实验室筛选所得。本发明具有显著改善NAFLD组织病变并抑制脂滴形成的地衣芽孢杆菌BL3 Sir2样蛋白经基因克隆于大肠杆菌表达纯化制备,实验结果表明其可以通过调节肝细胞脂代谢相关基因并影响PPAR-γ和PGC-1α通路,调控与脂代谢、氧化应激和胰岛素抵抗等过程相关的蛋白水平,从而减轻肝组织内的脂质蓄积,改善NAFLD大鼠的代谢异常,为益生菌Sir2样蛋白用于预防和治疗NAFLD提供一种新的治疗策略,以用于NAFLD防治药物或食品中的应用。(Bacillus licheniformis BL3Sir 2-like protein for improving NAFLD tissue lesion and application thereof. The invention discloses a bacillus licheniformis BL3Sir 2-like protein capable of remarkably improving non-alcoholic fatty liver disease tissue lesion and inhibiting lipid droplet formation and application thereof, belonging to the technical field of biology. The bacillus licheniformis BL3Sir 2-like protein for obviously improving NAFLD tissue lesion and inhibiting lipid droplet formation is prepared from Baclicuslicheniformis BL3 strain, which was screened in this laboratory. The invention can obviously improve NAFLD tissue lesionThe bacillus licheniformis BL3Sir 2-like protein for inhibiting lipid droplet formation is prepared by gene cloning in escherichia coli expression and purification, and experimental results show that the protein can regulate the protein level related to processes such as lipid metabolism, oxidative stress, insulin resistance and the like by regulating related genes of hepatocyte lipid metabolism and influencing PPAR-gamma and PGC-1 alpha pathways, thereby relieving lipid accumulation in liver tissues, improving the metabolic abnormality of NAFLD rats, and providing a new treatment strategy for the probiotic Sir 2-like protein for preventing and treating NAFLD, so as to be applied to NAFLD prevention and treatment medicines or foods.)

1. A Bacillus licheniformis BL3Sir 2-like protein capable of remarkably improving nonalcoholic fatty liver disease tissue lesion and inhibiting lipid droplet formation is characterized in that the Bacillus licheniformis BL3Sir 2-like protein is an NAD-dependent protein⁺The deacetylase of (1).

2. The use of the Bacillus licheniformis BL3Sir 2-like protein as claimed in claim 1 for significantly improving non-alcoholic fatty liver disease tissue pathology and inhibiting lipid droplet formation in the preparation of a medicament for the treatment of non-alcoholic fatty liver disease, lipid metabolism disorder or metabolic syndrome.

3. A food for improving non-alcoholic fatty liver disease tissue lesion, which comprises an effective dose of the Bacillus licheniformis BL3Sir 2-like protein component as an active ingredient, wherein the Bacillus licheniformis BL3Sir 2-like protein component can significantly improve non-alcoholic fatty liver disease tissue lesion and inhibit lipid droplet formation.

4. A medicament for preventing or treating non-alcoholic fatty liver disease tissue lesion, which comprises an effective dose of the bacillus licheniformis BL3Sir 2-like protein component with the effects of significantly improving non-alcoholic fatty liver disease tissue lesion and inhibiting lipid droplet formation according to claim 1 as an active ingredient, and a pharmaceutically acceptable carrier.

5. A food product for preventing lipid metabolism disorders, which comprises an effective amount of the Bacillus licheniformis BL3Sir 2-like protein ingredient according to claim 1 as an active ingredient, which significantly improves non-alcoholic fatty liver disease histopathology and inhibits lipid droplet formation.

6. A medicament for treating lipid metabolism disorder, which comprises an effective dose of the bacillus licheniformis BL3Sir 2-like protein component with the effect of significantly improving nonalcoholic fatty liver disease histopathology and inhibiting lipid droplet formation according to claim 1 as active ingredient, and a pharmaceutically acceptable carrier.

7. A food product for preventing metabolic syndrome, comprising an effective amount of the Bacillus licheniformis BL3Sir 2-like protein ingredient with effect of significantly improving nonalcoholic fatty liver disease histopathology and inhibiting lipid droplet formation according to claim 1 as an active ingredient.

8. A medicament for preventing and treating metabolic syndrome, which comprises an effective dose of the bacillus licheniformis BL3Sir 2-like protein component with the effects of significantly improving nonalcoholic fatty liver disease tissue lesion and inhibiting lipid droplet formation according to claim 1 as an active ingredient, and a pharmaceutically acceptable carrier.

Technical Field

The invention belongs to the field of new mechanisms for treating diseases by using probiotic Sir2 protein, and particularly relates to a bacillus licheniformis BL3Sir 2-like protein capable of remarkably improving non-alcoholic fatty liver disease histopathological changes and inhibiting lipid droplet formation and application thereof.

Background

Sir2 (Silene information regulator) protein is mainly located in nucleolus of cell, and is a highly conserved NAD⁺Dependent protein deacetylases, are present in bacteria and mammals. Sir2 protein with NAD⁺Influencing each other, on the one hand, the regulation of Sir2 protein function is mediated by NAD⁺Influence of synthetic and salvage pathways, in particular deacetylation of Sir2 protein requires NAD⁺As a cofactor; on the other hand, NAD⁺Intermediates of the salvage pathway, such as nicotine and nicotinic acid, with NADH and some NAD⁺Metabolites may also modulate the activity of Sir2 protein. Numerous studies have now shown that the mammalian Sirtuin family of proteins are involved in regulating cellular metabolism and help to delay, or even arrest, metabolic-related diseases.

Related researches show that Sirtuins are widely involved in regulating body energy metabolism and maintaining body glycolipid metabolism steady state, wherein the Sirtuins are researched most widely as SIRT1, SIRT3 and SIRT 6. SIRT1, the most studied member of the family, has been shown to regulate not only deacetylation of histones, but also non-histones, by current studies. Research shows that the function of SIRT1 in improving mitochondrial dysfunction is realized by regulating a SIRT1-SIRT 3-mitochondrial pathway, and the high-sugar-induced insulin resistance of skeletal muscle cells is improved when SIRT1 is over-expressed. In muscle, however, SIRT1 is able to enhance fatty acid oxidation in mitochondria by deacetylating PGC 1-a. In addition, SIRT3 is one of the most prominent mitochondrial deacetylases, many of which have been identified, many of which are involved in regulating important metabolic balances. Such as SIRT3 deacetylation to activate isocitrate dehydrogenase 2, which participates in the tricarboxylic acid cycle; SIRT3 also activates glutamate dehydrogenase in the tricarboxylic acid cycle, although the physiological significance of glutamate dehydrogenase deacetylation is not clear. Furthermore, SIRT3 deacetylated complexes I, II, III are involved in oxidative phosphorylation, the final phase of mitochondrial aerobic respiration. Whereas overexpression of mitochondrial Sirtuin proteins in HEK293T cells altered glycolysis and mitochondrial function. Second, SIRT6 is involved in the stabilization and reparative metabolism of genomic DNA and in senescence. It has been shown that SIRT6 deletion causes hypoglycemia in mice, whereas SIRT6 deletion increases glucose uptake in cells and glycolysis increases leading to decreased mitochondrial respiratory function. Thus, Sirtuin proteins are closely related to cellular energy, lipid metabolism, and to some extent serve as switches from glycolysis to oxidative metabolism.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases, the incidence rate is rising year by year, and at present, about 10 hundred million people are affected worldwide without specific drugs and treatment methods. Although numerous scientists have conducted extensive research on NAFLD, the pathogenesis of NAFLD is still unclear, and the cause is quite complex and is regulated by various factors, such as insulin resistance, oxidative stress, and disorders of adipocyte energy metabolism. Therefore, therapeutic means for reducing the incidence of NAFLD from the source by improving the disorder of hepatocyte energy metabolism, anti-oxidative stress, and inhibiting insulin resistance, etc. have become the focus of research in the present stage. There have been a number of reports in the literature that Sir 2-like deacetylase proteins play a key role in the amelioration and treatment of the progression of metabolic diseases such as NAFLD, and these studies have focused primarily on SIRT1, SIRT3 and SIRT6 mentioned earlier.

More and more studies indicate that probiotics can improve diseases such as metabolism. The probiotics have Sir2 homologous proteins, but few reports about the biological functions of the Sir2 protein in the probiotics. In recent years, the change of intestinal flora is found to have very important relevance to the occurrence of NAFLD, and researches show that probiotics are directly connected with gastrointestinal tract infection, bacterial translocation, cholesterol metabolism and the like. Bacillus licheniformis (Bacillus licheniformis) Is gram-positive (G)⁺) Facultative anaerobic, sporulation, probiotic, commonly known as whole intestine, is one of the existing speciesThe strain with multiple special functions can produce various enzymes, proteins and other substances, and has extremely wide research value in the aspects of animal husbandry, agriculture, medicine, food and the like. The bacillus licheniformis has the function of preventing and treating diseases by using a live bacterial preparation and has no harm to organisms. Therefore, the antibacterial agent can be used for achieving the purposes of preventing and controlling bacteria and improving the health level by utilizing the antagonistic action of the antibacterial agent on the bacteria and the biological antagonistic action of the bacteria, which are harmless to the human body and even promote the antagonistic action of foreign bacteria or excessive bacteria in the human body. Because the bacillus licheniformis is a facultative anaerobe, the bacillus licheniformis can play a role of biological oxygen deprivation, so that the probiotics can obtain a better growth environment and maintain the ecological balance in intestinal tracts.

Current studies have shown that the mammalian Sirtuin family of proteins can regulate cellular metabolism, including cellular respiration, fatty acid metabolism, and the like. Maintenance of normal Sirtuins activity helps to delay, or even prevent, the development of metabolic-related diseases. Previous studies on the mammalian Sirtuin protein family show that it is widely involved in regulation of glycolipid metabolism and maintenance of energy metabolism balance, and is expected to be a target for metabolic diseases. However, for the current research, few reports are made about the biological functions of the homologous proteins of the probiotic Sir2, and the biological functions are still in the initial stage, and especially the regulation functions in the aspect of metabolism are much less researched. NAFLD, which is found to be induced by metabolic disorders or the like, has not been a specific drug or therapeutic strategy so far. The probiotic Bacillus licheniformis in the present study also containssir2The homologous genes of (1) have Sir 2-like proteins, and although the SIRT1, the SIRT3 and the SIRT6 of the mammalian Sirtuin family can improve NAFLD, the regulation effect of prokaryotic Sir2 protein on NAFLD is not reported, and no related research on the influence of the Bacillus licheniformis BL3Sir2 protein on lipid metabolism in cells is carried out, so that the genes are worthy of intensive research.

Disclosure of Invention

According to the invention, the Bacillus licheniformis BL3Sir 2-like protein which can obviously improve the tissue pathological changes of the non-alcoholic fatty liver diseases and inhibit the formation of lipid droplets is screened out, and the treatment action mechanism of the protein is researched, so that the protein plays a role in treating diseases.

The method for constructing the rat model induces the rat to generate the non-alcoholic fatty liver disease by adopting a method of combining high-fat feed with carbon tetrachloride injection, and carries out intervention treatment by using purified bacillus licheniformis BL3Sir 2-like protein.

According to the invention, the bacillus licheniformis BL3Sir 2-like protein is used for carrying out intervention treatment on a rat model, and the influence of BL3Sir 2-like protein on the liver tissue morphology, pathological changes, serum liver function indexes, hepatocyte lipid metabolism related gene expression, PPAR-gamma, PGC-1 alpha and downstream target gene expression of the PPAR-gamma and PGC-1 alpha is researched.

The bacillus licheniformis BL3Sir 2-like protein provided by the invention can obviously improve the tissue lesion of NAFLD, inhibit lipid droplet formation and reduce the contents of alanine aminotransferase, aspartate aminotransferase, cholesterol and triglyceride in serum, thereby protecting the liver function of NAFLD rats.

The bacillus licheniformis BL3Sir 2-like protein provided by the invention can regulate the protein level related to processes such as lipid metabolism, oxidative stress, insulin resistance and the like by regulating liver cell lipid metabolism related genes and influencing PPAR-gamma and PGC-1 alpha pathways, thereby reducing lipid accumulation in liver tissues and improving the metabolic abnormality of NAFLD rats.

The invention provides a bacillus licheniformis BL3Sir 2-like protein which can obviously improve the tissue lesion of non-alcoholic fatty liver disease and inhibit the formation of lipid droplets and can be used for preparing a medicament for treating non-alcoholic fatty liver disease, lipid metabolism disorder or metabolic syndrome.

Compared with the prior art, experiments show that the Bacillus licheniformis BL3Sir 2-like protein which can obviously improve nonalcoholic fatty liver disease tissue lesion and inhibit lipid droplet formation can protect the liver function of a NAFLD rat, regulate and control the protein level related to the processes of lipid metabolism, oxidative stress, insulin resistance and the like by regulating liver cell lipid metabolism related genes and influencing PPAR-gamma and PGC-1 alpha pathways, so that the lipid accumulation in the liver tissue is relieved, the metabolic abnormality of the NAFLD rat is improved, and a novel treatment strategy is provided for the probiotic Sir2 protein to prevent and treat the NAFLD, so that the probiotic Sir2 protein can be applied to the application of NAFLD prevention and treatment medicines or foods.

Drawings

Figure 1 is a graph of liver morphology in example 1 NAFLD rats with different treatments.

Figure 2 is the liver histopathological HE staining (100 fold) of example 1 NAFLD rats with different treatments.

FIG. 3 is ALT and AST levels in serum of example 1 NAFLD rats, where the values are shown as mean. + -. standard error; p < 0.05 and p < 0.01 compared to model group; n = 8.

FIG. 4 is the TC and TG levels in serum of example 1 NAFLD rats, where the values are shown as mean. + -. standard error; p < 0.05 and p < 0.01 compared to model group; n = 8.

FIG. 5 is the mRNA expression level of the gene involved in the regulation of lipid metabolism in rat hepatocytes of example 1 NAFLD, where the values are shown as mean. + -. standard error; p < 0.05 and p < 0.01 compared to model group; n = 8.

FIG. 6 is a graph of expression of protein related to lipid metabolism regulation of NAFLD rat hepatocytes detected by Western blot in example 1.

FIG. 7 is the expression level of the protein involved in regulating lipid metabolism of rat hepatocytes of example 1 NAFLD, where the values are shown as mean. + -. standard error; p < 0.05 and p < 0.01 compared to model group; n = 8.

FIG. 8 is the mRNA levels of PPAR- γ and PGC-1 α and genes downstream thereof of example 1, where the values are shown as means. + -. standard error; p < 0.05 and p < 0.01 compared to model group; n = 8.

FIG. 9 is a diagram of Western blot detection of PPAR-gamma and PGC-1 alpha and their downstream protein expression in example 1.

FIG. 10 is the levels of PPAR- γ and PGC-1 α and their downstream proteins of example 1, where the values are shown as means. + -. standard error; p < 0.05 and p < 0.01 compared to model group; n = 8.

Detailed Description

The present invention will be described in further detail with reference to examples.