CN111840331B - Application of bear gall exosome in preparation of medicine for treating type II diabetes - Google Patents

Abstract

The invention discloses an application of bear gall exosomes in preparing a medicine for treating type II diabetes, and belongs to the field of molecular biology. The bear gall exosome contains multiple miRNA effective components which effectively act on type II diabetes, can reduce the blood sugar of rats and improve related symptoms, and provides a new direction for preparing a medicament for treating diabetes; the bear gall exosome applied to preparing the medicament for treating the type II diabetes is simple and easy to obtain, can be used independently or in the form of a medicinal composition, has a wide range, and is economical and feasible.

Description

Application of bear gall exosome in preparation of medicine for treating type II diabetes

Technical Field

The invention relates to the technical field of molecular biology, in particular to application of bear gall exosomes in preparation of a medicine for treating type II diabetes.

Background

Diabetes Mellitus (DM) is a metabolic disease characterized by hyperglycemia. Hyperglycemia is primarily due to a defect in insulin secretion or an impaired biological action thereof, or both. Chronic hyperglycemia can lead to chronic damage to, and dysfunction of, various tissues, particularly the eye, kidney, heart, blood vessels, and nerves. The modern times show that the diabetes is too big, and no effective radical cure mode and medicine exist.

The clinical diabetes treating medicine can only lower blood sugar, but has poor curative effect and causes great damage to the body, such as insulin and metformin. Diabetes mellitus is a disease of a chronic glycolipid metabolic disorder formed by multi-factor interaction, and the diabetes mellitus itself and related complications seriously affect the global human health. Diabetic complications are poorly detected at the early stages and often develop before diabetes, i.e., impaired Glucose Tolerance (IGT). Various oral hypoglycemic agents for treating diabetes, such as biguanides, sulfonylureas, thiazolidinediones and the like, cannot effectively prevent and treat diabetic complications.

The traditional Chinese medicine has the characteristics of multiple components, multiple ways, multiple targets and integral regulation, and has unique advantages in the prevention and treatment of diabetes and complications thereof. Bear gall is one of four major precious animal drugs, is the dry gall bladder (bile) of animals in the family of bear, and is known as 'gold in medicine'. The traditional Chinese medicine is obtained by living bile drainage and a freeze-drying method at present, and basically has no damage to bears. It is recorded in the treatise of the treatise on the theory of drug Property for "treating infant malnutrition, killing parasites and malignant boil"; the calendar-code medical books record the effects of cooling liver blood, purging liver fire, clearing heat and removing toxicity, clearing liver and improving vision and the like, and the traditional Chinese medicine has exact curative effect for thousands of years. Bear gall as a medicine is recorded in 'Xin Xiu Ben Cao' of the Tang Dynasty, and has a history of thousands, three and hundreds years to date. The compendium of materia medica of the Ming Dynasty Li Shizhen also carries: bear gall, bitter and cold in smell, non-toxic and enters liver, gall and heart meridians. Modern pharmaceutical research proves that bear gall has the effects of clearing away heat and toxic materials, soothing liver and benefiting gallbladder, removing nebula and improving eyesight, and relieving spasm and pain. The pharmacological activities of bear gall such as anti-inflammation, liver protection, analgesia and the like are also found. Exosomes refer to small membrane vesicles (30-150 nm) containing complex RNAs (such as microRNAs or miRNA, miR and the like) and proteins in cells, and refer to disc-shaped vesicles with diameters of 40-100nm, which play an important role in cell-to-cell communication. At present, exosomes are found to have stronger pharmacological activity and better treatment effect than general chemical components.

Therefore, it is necessary to research the bear gall exosomes, and a new research direction is provided for preparing the medicine for treating diabetes by extracting and analyzing effective active ingredients, so as to provide support for the application of the bear gall exosomes in preparing the medicine for treating diabetes.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of bear gall exosomes in preparation of a medicament for treating type II diabetes, and provide a new direction for preparation of a medicament for treating diabetes.

The invention provides an application of bear gall exosomes in preparing a medicine for treating type II diabetes, wherein the bear gall exosomes comprise one or more of the following miRNAs: rno-miR-592, rno-miR-675-5p, rno-miR-1247-3p, rno-miR-3072, rno-miR-3541, rno-miR-343, rno-miR-673-3p, rno-miR-6324, rno-miR-1247-5p, rno-miR-370-3p, rno-miR-3585-5p, rno-miR-375-5p, rno-miR-92a-1-5p, rno-miR-434-5p, rno-miR-668, rno-miR-9a-5p rno-miR-455-5p, rno-miR-3084a-3p, microRNA-368, microRNA-132-3p, microRNA-181a, microRNA-30b, microRNA-192, microRNA-29c, microRNA-149-3p, microRNA-144, microRNA-142, microRNA-Let-7b, microRNA-130b, microRNA-9-5p, microRNA-23b, microRNA-21, microRNA-20, microRNA-126.

Further, the bear bile exosomes comprise one or more of the following mirnas: rno-miR-592, rno-miR-675-5p, rno-miR-1247-3p, rno-miR-3072, rno-miR-3541, rno-miR-343, rno-miR-673-3p, rno-miR-6324, rno-miR-1247-5p, rno-miR-370-3p, rno-miR-3585-5p, rno-miR-375-5p, rno-miR-92a-1-5p, rno-miR-434-5p, rno-miR-668, rno-miR-9a-5p, rno-miR-455-5p and rno-miR-3084a-3p.

The invention also aims to provide a pharmaceutical composition for treating type II diabetes, which comprises the bear gall exosome in the application.

Further, the pharmaceutical composition comprises, as an active ingredient, bear's gall;

further, the pharmaceutical composition comprises a pharmaceutically acceptable carrier as an active ingredient;

further, the pharmaceutical composition comprises pharmaceutically acceptable auxiliary materials as active ingredients.

Compared with the prior art, the invention has the beneficial effects that:

1) The invention provides an application of bear gall exosomes in preparing a medicament for treating type II diabetes, wherein the exosomes comprise multiple bear gall exosome miRNA, can effectively reduce blood sugar and related symptoms of type II diabetes rats, and provides a new direction for preparing the medicament for treating diabetes;

2) The bear gall exosome applied to preparing the medicine for treating the type II diabetes is simple and easy to obtain, can be used independently or in the form of a medicine composition, has wide range, and is economical and feasible.

Drawings

FIG. 1A shows the size distribution of exosomes of bear gall powder; FIG. 1B is a transmission electron microscope image of bear gall powder exosome particles; and 1C is the HPLC result of the bear gall powder exosome, the bear gall powder and the ursodeoxycholic acid.

FIG. 2 is the clinical signs of type II diabetic rats administered with bear bile exosomes.

FIG. 3 shows the results of the blood glucose change and glucose tolerance tests of type II diabetic model rats administered with bear gall exosomes.

FIG. 4 is organ index of type II diabetes model rats after administration of bear gall exosomes.

FIG. 5 shows the ALT and AST levels in serum of type II diabetes model rats.

FIG. 6 is the levels of SOD, MDA and GSH in the liver of type II diabetes model rats.

FIG. 7 shows the histological analysis of liver and pancreas of type II diabetes model rats.

FIG. 8 shows the expression of TNF-. Alpha.and IL-1. Beta. In the liver of type II diabetes model rat.

FIG. 9 shows the levels of PPAR α, NF- κ B and CTP7A1 in the liver of type II diabetes model rats.

FIG. 10 is a diagram of the regulation of glycolipid metabolism by bear gall exosomes.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.

The present invention is based on the unexpected finding that: the bear gall and the bear gall exosome components can reduce the blood sugar and the related disease damage of diabetic mice, so the bear gall and the bear gall exosome components can be used as the preventive and therapeutic drugs for diabetes.

As used herein, the exosome components of the present invention have the following characteristics (fig. 1A):

as known to those of ordinary skill in the art, the above exosomes have the following particle size distributions, respectively (fig. 1B):

the bear gall (in particular to Xiong Yinliu bile freeze-dried powder, namely bear gall powder) can be purchased from Lu Ye Ke pharmaceutical Co., ltd, etc. of Sichuan province through a commercial way, and then is separated strictly according to a standard exosome particle separation method (differential centrifugation method) in related documents, and the purity and related physical and chemical characteristics of the bear gall accord with related exosome standards.

The bear gall exosome of the present invention may be used alone or in the form of a pharmaceutical composition. The pharmaceutical composition comprises the exosome component of the invention as an active ingredient, bear gall and a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition of the present invention comprises 0.1 to 99.9% by weight of the exosome component of the present invention and bear's gall as active ingredients. The medicinal carrier does not destroy the pharmaceutical activity of the bile acid component and the bear gall, and simultaneously ensures the effective dosage of the bear gall, and the dosage of the bear gall when the better medicinal carrier is used is nontoxic to human bodies.

Such pharmaceutically acceptable carriers include, but are not limited to: lecithin, aluminum stearate, alumina, ion exchange materials, self-emulsifying drug delivery systems, tweens or other surfactants, serum proteins, buffer substances such as phosphates, glycine, sorbic acid, water, salts, electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, magnesium silicate, mixtures of saturated fatty acid partial glycerides, and the like.

Other conventional pharmaceutical adjuvants such as binder (e.g. microcrystalline cellulose), filler (e.g. starch, glucose, anhydrous lactose and lactose beadlets), disintegrant (e.g. crosslinked PVP, sodium crosslinked carboxymethyl starch, sodium crosslinked carboxymethyl cellulose, low-substituted hydroxypropyl cellulose), lubricant (e.g. magnesium stearate), and absorption enhancer, adsorption carrier, flavoring agent, sweetening agent, excipient, diluent, wetting agent, etc.

The bear gall exosome and the pharmaceutical composition thereof can be prepared according to the conventional method in the field and can be administrated by an intestinal or parenteral or local route. The oral preparation comprises capsule, tablet, oral liquid, granule, pill, powder, pellet, and unguent; parenteral preparations include injections and the like; topical preparations include creams, patches, ointments, sprays, and the like. Oral preparations and injections are preferred.

The administration route of the bear gall exosome and the pharmaceutical composition thereof can be oral administration, sublingual administration, transdermal administration, intramuscular administration or subcutaneous administration, skin mucosa administration, vein administration, urethra administration, vagina administration and the like.

Examples

1. Preparation of bear gall exosome medicine

The purchased bear gall powder (source: chlorophycoac pharmaceutical Co., ltd., sichuan province, batch No.: 20190101) was taken and separated according to the relevant literature method (differential low temperature centrifugation). Taking a proper amount of distilled water to dissolve the bear gall powder, firstly centrifuging for 20min at 3000g/min, then centrifuging for 15min at 5000g/min, taking the solution again centrifuging for 40min at 10000g/min, taking the solution again centrifuging for 60min at 100000g/min, collecting precipitates to obtain bear gall exosomes, and observing the surface of the bear gall exosomes and measuring the particle size distribution of the bear gall exosomes by using a transmission electron microscope and a Brookhaven BI-200SM laser scattering spectrometer. After meeting the standard, the raw materials are stored at the temperature of minus 80 ℃ for standby, and when the raw materials are used, a proper amount of distilled water is taken to be dissolved to a specified concentration.

Wherein: the particle size distribution of the bear gall exosomes is shown in figure 1A, the transmission electron microscope image of the bear gall powder exosome particles is shown in figure 1B, and the chemical fingerprint (HPLC) of the bear gall powder exosome particles is shown in figure 1C, and the samples are ursodeoxycholic acid (XQY), exosomes (WMT) and bear gall powder (XDF), and the results show that the exosomes do not contain various original chemical components of the bear gall powder basically. (wherein each peak in the HPLC chart of the bear bile powder represents 1, cholic acid; 2, taurodeoxycholic acid; 3, ursodeoxycholic acid; 4, sodium cholate; 5, taurocholic acid; 6, hyodeoxycholic acid; 7, chenodeoxycholic acid; 8, deoxycholic acid; 9 taurodeoxycholic acid).

As can be seen from FIG. 1, the appearance of the bear gall exosomes obtained by differential centrifugation meets the characteristics of the related exosomes, and the bear gall exosomes mainly comprise microparticles with the particle size of 200-500nm, which belong to the range of the particle size of the exosomes. HPLC results show that components in the bear gall exosomes do not absorb ultraviolet rays, and main contents of the bear gall exosomes can be determined to comprise various microRNAs.

The main chemical components and contents of bear gall powder are shown in table 1.

Table 1:

2. establishment and administration of type II diabetes rat model

200-220g male Sprague Dawley (SD) rats were purchased, adaptively bred for 3 days, and then randomly divided into a blank control group, a model group, a metformin group (MET, 200 mg/kg), and a bear bile exosome group (high-dose, 40 mg/kg), 10 animals per group. The rats in the blank group are normally raised in 2 cages each, and the rats in other groups are singly raised in single cage. The experiment was carried out by feeding the rats with high-sugar and high-fat diet for 8 weeks, feeding streptozotocin (STZ, 40 mg/kg) by intraperitoneal injection after fasting for 12 hours, and feeding the rats with high-sugar and high-fat diet for 7 days, and taking a model with blood sugar higher than 16.0mmol/L as a diabetic rat model. Each administration group was intragastrically administered according to the same phase Guan Jiliang, and the blank group and the model group were administered with the same amount of physiological saline, and the rats were sacrificed in a sterile environment after 6 weeks of continuous administration. The livers were immediately stored in liquid nitrogen and the levels of all relevant mirnas in liver tissue were determined. Clinical sign comparison of rats is shown in Table 2, and clinical sign expression of type II diabetes model rats after administration of bear gall exosome (40 mg/kg) is shown in FIG. 2A, and body weight expression is shown in FIG. 2B.

As can be seen from FIG. 2, the signs (2A) and the body weight (2B) of the model group are obviously worse than those of the normal control group, and all rats in the model group have obvious diabetic symptoms, so that the model building is successful. After the low, medium and high dose groups of the metformin and bear gall exosomes are given, the physical signs are obviously changed, compared with a model group, the physical signs are obviously improved and are closer to a blank group, wherein the medium dose group and the high dose of the bear gall exosomes have lower drug effects and are better than the metformin group.

Table 2:

3. determination of blood sugar lowering effect of bear gall powder exosome

Blood glucose and body weight of rats in each group were measured on time starting from day 1 of week 1 of administration, and changes in signs of rats were observed and recorded for six weeks. On day 5 of the 6 th week of administration, overnight fasted rats were subjected to the Oral Glucose Tolerance Test (OGTT). Each group of ratsOrally administering glucose at a dose of 2g and kg ^-1 . Blood glucose was measured directly from the tail vein using a one-touch glucometer at 0, 30, 60, 90 and 120 minutes.

As can be seen from FIG. 3A, the blood sugar of the model group is significantly increased (p < 0.05) compared with that of the normal control group, and the blood sugar of the model group rats is higher than 16.0, so that the modeling is successful. After the metformin and the bear gall exosome are administered to the low, medium and high dose groups, the blood sugar of the rats is obviously changed (p is less than 0.05), and the blood sugar is obviously reduced compared with the model group (p is less than 0.05), wherein the drug effect of the bear gall exosome is better than that of the positive group. As can be seen from fig. 3A, FBG levels in the model group were significantly higher than those in the NC group, and FBG in the rat was significantly reduced after treatment with bear gall exosomes (p < 0.05).

Oral Glucose Tolerance Test (OGTT) test (3B). The area under the glucose curve of the model group was significantly increased compared to the blank group (p < 0.05); the blood glucose levels in the blank group decreased to the initial level after 120 minutes, while the model group remained at a higher level. Treatment with bear gall exosomes in a diabetic rat model significantly reduced the rise in blood glucose levels after oral glucose administration. These results indicate that bear bile exosomes can lower blood glucose and improve glucose metabolism in T2DM-NAFLD rats.

4. Determination of metabolism regulating effect of bear gall powder exosome

On day 3 of the 6 week period of dosing, each rat in this experiment was placed in a metabolic cage to collect the amount of feces and urine for 12 hours, weighed and compared for metabolic capacity. Stool and urine changes for the T2DM rat model 12h are shown in table 3.

Table 3:

note: in Table 3, compared with the normal control group, ^## p<0.01， ^### p <0.001; compared with model group< 0.05，**p < 0.01， ***p < 0.001。

As can be seen from Table 3, the urine and stool amount of the model group rats is significantly increased (p < 0.05) compared with that of the normal control group, and the sugar content in urine is higher, which proves that the model group rats have significant diabetes characteristics and the model of diabetes is successfully modeled. The metabolic capacity after the administration of metformin and bear gall exosomes is obviously improved (p is less than 0.05), wherein the bear gall exosomes are better than the positive group.

5. Determination of organ index of bear gall powder exosome

After sacrifice of the drug administration for 6 weeks, liver, lung, spleen, brain and kidney organs were weighed and the relative organ coefficients were calculated.

The formula: organ factor = (organ weight (g) × 1000 × 100%)/rat body weight (g)

As shown in fig. 4A, 4B and 4C, the liver, spleen and kidney organ indexes of the model group rats were significantly changed, respectively, wherein the liver and kidney indexes were significantly increased (p < 0.05), the spleen index was significantly decreased (p < 0.05), which indicates that the liver and kidney of the rats had serious lesions, and the immune organs of the rats were unable to repair the self-disease deterioration. After the metformin and the low, medium and high dose groups of bear gall exosomes are administered, the liver and kidney indexes of rats are remarkably reduced (p is less than 0.05), the spleen index is remarkably increased (p is less than 0.05), and the medium dose group and the high dose group of the bear gall exosomes have better drug effects and the positive group.

6. Measuring the level of four items of blood fat in serum

The measurement is carried out strictly according to the requirements of TC, TG, HLD-C and LDL-C kits (Nanjing Biotechnology Co., ltd.).

The fourth term of blood lipid is the conventional index of T2DM, which reflects the severity of pathological development of T2 DM. To investigate the effect of WMT on improvement of serum lipids in T2DM rats, lipids in T2DM rats were determined (as shown in table 4). Compared with the NC group, the TC, TG, LDL-c and FFA contents of the T2DM group are obviously increased (p < 0.01), and the HDL-c of the T2DM group is obviously reduced (p < 0.001). After 6 weeks of treatment, the rats in the treatment group all had significantly reduced TC, TG, LDL-c levels (p < 0.05) and significantly increased HDL-c levels (p < 0.01), with the exosomes having the best potency. This suggests that WMT may act to lower blood glucose by modulating TC, TG, LDL-c, HDL-c levels.

Table 4:

note that in Table 4, compared with the normal group, ^## p<0.01， ^### p <0.001; comparison with model group< 0.05，**p < 0.01， ***p < 0.001。

7. Determination of ALT and AST levels in serum

When the liver of a body is injured, cells secrete a large amount of ALT and AST to enter blood, and with the diffusion of ALT and AST, the body can repair the injured liver by self, and if ALT and AST cannot be repaired, ALT and AST are accumulated all the time. We determined ALT and AST levels in serum from T2DM rats (shown in FIGS. 5A and 5B, respectively, where compared to normal groups) ^## p<0.05， ^## p<0.01， ^### p <0.001; comparison with model group< 0.05，**p < 0.01，***p <0.001 We found significant elevations (p) of ALT and AST in model group rats<0.01 3 times that of normal rats, which is a very dangerous signal; after 6 weeks of treatment, ALT and AST levels were significantly reduced in all treated groups (p)<0.05). This suggests that WMT may play a hepatoprotective role by regulating ALT and AST levels.

8. Determination of SOD, MDA and GSH levels in the liver

Oxidative stress is now considered to be the causative mechanism of diabetes and liver damage, and antioxidant enzymes play an important role in maintaining the renal oxidative balance. If oxidative stress is not improved in a timely manner, severe inflammation, even cirrhosis and canceration, may result. T-SOD and GSH-Px are key antioxidants for scavenging free radicals. We examined the levels of T-SOD, MDA and GSH-Px in rat liver in order to investigate the pharmacodynamic mechanism of WMT (shown in FIGS. 6A, 6B and 6C, respectively, note that, in comparison with the normal group, ^## p<0.05， ^## p<0.01， ^### p <0.001 (ii) a In comparison with model group< 0.05，**p < 0.01， ***p <0.001). SOD was significantly reduced in the model group (p) compared to the blank group<0.01 Whereas MDA and GSH are significantly elevated (p)<0.01 This) ofSuggesting severe oxidative stress in liver tissue; after 6 weeks of treatment, SOD in each of the administered groups was significantly increased (p) as compared to the model group<0.01 And the levels of MDA and GSH are significantly reduced (p)<0.01). The result shows that WMT can regulate the oxidative stress of the liver of a T2DM rat. The above results suggest that oral administration of WMT may significantly improve the antioxidant capacity of the liver of T2DM rats and reduce DNA oxidative damage by modulating oxidative stress.

9. Histopathological observation of liver and pancreas

The histological analysis shown in FIG. 7 was obtained following the strict histopathological HE, oil red O and PAS staining procedures of the liver and pancreas.

Histological analysis of the pancreas showed (as shown in fig. 7A) that there were significant differences in the morphology of the islets and in the number of islets. In the NC group of rats, islets are clusters of round or oval cells, scattered in healthy acinar cells with clear borders. The number of islets was significantly reduced in the T2DM group compared to the NC group, and the former also showed irregular islet morphology and atrophy. In addition, pancreatic cells of group D rats exhibited degenerative changes such as nuclear contraction or lysis, cell necrosis and vacuolization. Rats treated with MET and WMT showed improvement in histological abnormalities, with markedly increased islet numbers, improved shape, and reduced cell damage, with only minor liver cell abnormalities seen with WMT.

The histological analysis of the liver showed (as shown in fig. 7B) the ordered structure of hepatocytes of NC group, with well-defined morphology and rounded shape, with nuclei located in the center. In contrast, the intracellular structure of rat hepatocytes in the T2DM group was extensively denatured and necrosed. After MET and WMT treatment, the structure and appearance pathology of hepatocytes was significantly improved.

The results of the liver oil red O staining analysis (as shown in fig. 7B) showed that there were no oil red drops in the liver tissue of the rats in the blank group, whereas the hepatocytes of the T2DM rats exhibited a large amount of deposited fat stained red. MET and WMT lipid deposition were significantly reduced compared to the model group, with only a small amount of lipid deposition seen with WMT.

The results of the liver PAS staining analysis (as shown in fig. 7B) showed that there was no accumulation of purple-colored glycogen in the hepatocytes of the blank rat, whereas the hepatocytes of the T2DM rat exhibited a large amount of glycogen accumulation stained purplish red. MET and WMT purple color glycogen accumulation was significantly reduced compared to the model group, with WMT showing only a small amount of purple color glycogen accumulation.

10. Determination of IL-1 beta and TNF-alpha levels in the liver

The assay was performed exactly according to the ELISA kit for IL-1. Beta. And TNF-. Alpha. (Wuhan cloud technologies, inc.).

Several studies have shown that TNF- α and IL-1 β are closely associated with liver damage in T2 DM. Thus, the expression of TNF-. Alpha.and IL-1. Beta.in rat liver was determined herein (as shown in FIG. 8, compared to the normal group, ^## p<0.05， ^## p<0.01， ^### p <0.001 v; comparison with model group, # p< 0.05， **p < 0.01， ***p <0.001. ) Significantly elevated levels of TNF-alpha and IL-1 beta in the liver (P) compared to the T2DM model mouse<0.001 After 6 weeks of treatment, the levels of TNF-alpha and IL-1 beta were significantly reduced (P) in the four treatment groups<0.001 In which the TNF-alpha and IL-1 beta levels of WMT decreased to a similar level as the blank, these experimental results show that oral administration of EE reduced the inflammatory response of the liver in T2DM rats.

11. Protein results for PPAR α, NF- κ B and CYP7A1

The assay was performed strictly in accordance with the protein assay requirements for PPAR α, NF- κ B and CYP7A 1.

The NF-. Kappa.B signaling pathway and PPAR. Alpha. Are involved in the pathogenesis and inflammation of T2DM liver injury. To investigate the potential mechanism of action of WMT on liver damage to T2DM, the protein levels of PPAR α, NF- κ B and CYP7A1 in the livers of T2DM and treated rats were determined (as shown in fig. 9). Compared with normal rats, the expression of PPAR alpha and NF-kappa B in the liver of the T2DM rat is obviously reduced (p is less than 0.05), and the expression of CYP7A1 is obviously reduced. After 6 weeks of treatment, the rat liver showed a significant decrease in PPAR α, NF- κ B expression (p < 0.05) and a significant increase in CYP7A1 expression (p < 0.05) compared to the T2DM group. This indicates that WMT has a significant regulatory effect on activation of hepatic PPAR α, NF- κ B and inhibition of CYP7 A1.

12. Analysis of effective components of bear gall exosome miRNA

In the experiment, the expression difference miRNA is found through genome sequencing comparison of a T2DM model mouse and a bear gall exosome high-dose group and is used as a component basis of the drug effect. The specific method comprises the following steps: sequencing the obtained original sequence by HiSeq, and filtering to obtain high-quality clean data. The small RNA sequences are classified and annotated to obtain the information of each component and miRNA expression quantity contained in the sample. Predicting a new miRNA for the small RNA segment which is not annotated; performing differential analysis, cluster analysis, target gene prediction and KEGG and GO function annotation on a target gene on the known miRNA and the new miRNA, and performing KEGG and GO enrichment analysis on the differential expression miRNA target gene.

The research results show that the bear gall exosomes meet the relevant standards of exosomes, and the diabetes symptoms of rats are obviously improved after the diabetes model rats are fed with various doses of bear gall powder exosomes. We found expression differential mirnas by gene sequencing comparison. rno-miR-592, rno-miR-675-5p, rno-miR-1247-3p, rno-miR-3072, rno-miR-3541, rno-miR-343, rno-miR-673-3p, rno-miR-6324, rno-miR-1247-5p, rno-miR-370-3p, rno-miR-3585-5p, rno-miR-375-5p, rno-miR-92a-1-5p, rno-miR-434-5p, rno-miR-668, rno-miR-9a-5p and rno-miR-455-5p, rno-miR-3084a-3P, rno-miR-RNA-368, rno-miR-RNA-132-3P, rno-miR-RNA-181a, rno-miR-RNA-30b, rno-miR-RNA-192, rno-miR-RNA-29c, rno-miR-RNA-149-3P, rno-miR-RNA-144, rno-miR-RNA-142, rno-miR-RNA-Let-7b, rno-miR-RNA-130b, rno-miR-RNA-9-5P, rno-miR-RNA-23b, rno-miR-RNA-2, rno-miR-RNA-20, rno-miR-RNA-126 and rno-miR-335 miRNA (P < 0.05) with significant difference is considered as a reliable and effective component of the fel Ursi exosomes. We enumerate the fold difference of the 35 mirnas in the liver of the diabetic animal model, as shown in table 5.

Table 5: fold difference of known miRNA pre-35 position in liver of diabetes animal model

Sequence of	Name of miRNA	T2DM vs WMT-high
			1	rno-miR-592	0.0000000051
2	rno-miR-675-5p	0.0000284951
			3	rno-miR-1247-3p	0.0000284951
4	rno-miR-3072	0.0000540168
			5	rno-miR-3541	0.0090001029
6	rno-miR-343	0.0109944439
			7	rno-miR-673-3p	0.0109944439
8	rno-miR-6324	0.0224891406
			9	rno-miR-1247-5p	0.0235420257
10	rno-miR-370-3p	0.0242250319
			11	rno-miR-3585-5p	0.0264052607
12	rno-miR-375-5p	0.0391660928
			13	rno-miR-92a-1-5p	0.0419618521
14	rno-miR-434-5p	0.0469993114
			15	rno-miR-668	0.0650204524
16	rno-miR-9a-5p	0.0766958698
			17	rno-miR-455-5p	0.0789450264
18	rno-miR-3084a-3p	0.0789450264
			19	rno-miR-RNA-368	0.1023875159
20	rno-miR-RNA-132-3p	0.1081944898
			21	rno-miR-RNA-181a	0.1218500666
22	rno-miR-RNA-30b	0.1305330019
			23	rno-miR-RNA-192	0.1512151758
24	rno-miR-RNA-29c	0.1794828776
			25	rno-miR-RNA-149-3p	0.1915808410
26	rno-miR-RNA-144	0.2460452597
			27	rno-miR-RNA-142	0.2460452597
28	rno-miR-RNA-Let-7b	0.2831877903
			29	rno-miR-RNA-130b	0.3186370107
30	rno-miR-RNA-9-5p	0.3345542382
			31	rno-miR-RNA-23b	0.3345542382
32	rno-miR-RNA-2	0.4064058104
			33	rno-miR-RNA-20	0.4064058104
34	rno-miR-RNA-126	0.4064058104
			35	rno-miR-335	0.4117625147

As seen from Table 5 fold differences, 35 miRNAs all showed differences, but in particular, 18 miRNAs such as rno-miR-592, rno-miR-675-5p, rno-miR-1247-3p, rno-miR-3072, rno-miR-3541, rno-miR-343, rno-miR-673-3p, rno-miR-6324, rno-miR-1247-5p, rno-miR-370-3p, rno-miR-3585-5p, rno-miR-375-5p, rno-miR-92a-1-5p, rno-miR-434-5p, rno-miR-668, rno-miR-9a-5p, rno-miR-455-5p, rno-miR-3084a-3p and the like have fold differences of less than 0.1, and are considered as important active ingredients.

The research results show that: the bear gall powder exosome can effectively reduce the blood sugar and relevant symptoms of type II diabetic rats, can regulate the comprehensive disorder state of glycolipid metabolism, and has good liver-protecting and bile-benefiting effects, so the bear gall powder exosome can be used as a medicament for preventing and treating diabetes.

The action principle is that the bear bile powder exosome contains miRNA with active ingredients, can play a role in protecting liver by regulating the levels of ALT and AST, can regulate the oxidative stress of the liver of a T2DM rat, improve the structure of hepatic cells, improve the structure of pancreatic islets, improve insulin sensitivity index, reduce fasting plasma glucose, improve glucose tolerance, reduce hepatic tissue lipid deposition, promote glycometabolism and transformation, has remarkable regulation effects on the activation of liver PPAR alpha and NF-kappa B and the inhibition of CYP7A1, plays roles in resisting inflammation, resisting oxidative stress and improving lipid metabolism, and thus plays roles in regulating carbohydrate ester metabolism, protecting liver and benefiting gallbladder.

More specifically: insulin resistance, oxidative stress and inflammation are key factors in the progression of NAFLD. ROS not only can damage proteins and DNA, but also in particular impair lipid homeostasis in hepatocytes, which is directly related to NAFLD. Cells produce antioxidant enzymes such as SOD, GSH-Px and CAT to reduce ROS. Typically, ROS produced by T2DM patients are formed by the autoxidation of glucose. The final product of lipid peroxidation is MDA, which is cytotoxic. SOD can catalyze the disproportionation of superoxide anion radicals, thereby modulating oxidative imbalances. GSH-Px reduces toxic peroxides to non-toxic hydroxyl compounds to protect the structure and function of cell membranes, the principle of which is shown in fig. 10. In the present invention, WMT was found to improve these changes in MDA, GSH-Px and SOD in T2DM liver. Chronic oxidative stress may affect the hepatocyte apoptosis and survival signals of T2DM by destroying hepatocyte structures, while WMT may suppress oxidative stress.

In addition, modern studies suggest that the pathogenesis of T2DM may be consistent with inflammation-related mechanisms; t2DM results in the release of a variety of pro-inflammatory factors (e.g., IL-1, IL-2, IL-6, and TNF- α) (as shown in FIG. 10), thereby promoting the development of liver inflammation and liver steatosis; PPAR α can regulate several biological processes caused by obesity, including inflammation and metabolism of lipids and glucose; activation of NF-. Kappa.B will regulate the amplification of many genes (e.g., CPY7A 1) and continue the inflammatory response; drugs can home in damaged liver and improve the microenvironment of the liver by modulating inflammation and immune homeostasis. The invention discovers that the levels of TNF-alpha and IL-1 beta in the liver of T2DM are increased, the levels of PPAR alpha and NF-kappa B are increased, and CYP7A1 is reduced. This suggests that the mechanism of efficacy of WMT may be involved in regulating oxidative imbalance and PPAR α/NF- κ B/CYP 7A1, thereby reducing inflammation of the liver. The invention considers that the pharmacological mechanisms of the three medicines are probably related to improving liver function, promoting the synthesis of glycogen from glucose and lactic acid, and enhancing the action and secretion of insulin, and the insulin promotes the conversion of glucose to glycogen, thereby reducing blood fat and blood sugar.

The invention is not limited to what has been described in the specification and embodiments, and additional advantages and modifications will readily occur to those skilled in the art, and it is therefore not intended to be limited to the specific details, representative embodiments, and illustrative examples shown and described herein, without departing from the spirit and scope of the general concept as defined by the appended claims and their equivalents.

Claims (4)

1. Application of fel Ursi exosome in preparing medicine for treating type II diabetes is provided.

2. The use according to claim 1, wherein the medicament for the treatment of type II diabetes comprises a pharmaceutically acceptable carrier.

3. The use according to claim 1, wherein the medicament for the treatment of type II diabetes comprises a pharmaceutically acceptable excipient.

4. The use as claimed in claim 1, wherein the pharmaceutical dosage form for the treatment of type II diabetes comprises any one of oral, parenteral and topical formulations.

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