Application of resveratrol in preparation of medicine for preventing or treating gallstone

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of resveratrol in preparation of a medicine for preventing or treating gallstone. The resveratrol can regulate liver cholesterol metabolism, reduce bile cholesterol saturation and relieve liver inflammation by up-regulating expression and functions of PPAR gamma in the liver, so that gall-stone formation is remarkably reduced, and related symptoms such as gallbladder dilatation and cholestasis are relieved. Therefore, the invention provides a new medicine for effectively preventing and treating the cholesterol gallstone, and the resveratrol provided by the invention has good clinical application value and application prospect as the medicine for preventing and treating the cholesterol gallstone. The medicine has the advantages of novel action target, reliable curative effect, no toxic or side effect, abundant resources and low price.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of resveratrol in preparing a medicine for preventing or treating gallstones. Background Art

[0002] Cholesterol gallstone disease is a prevalent disorder characterized by the formation of cholesterol-rich stones in the gallbladder, which has major public health implications worldwide. It is estimated that 10-15% of adults in Western populations suffer from gallstones, with higher incidences in women, obese individuals, and those with a history of diabetes or hyperlipidemia. The prevalence of cholesterol gallstone disease has also been increasing in Asian populations. This condition not only causes significant morbidity, but also imposes a considerable economic burden on healthcare systems due to the high costs associated with diagnosis, treatment, and ongoing management. In addition to these direct costs, the disease also creates an indirect economic burden through lost productivity and reduced quality of life for affected individuals, and may cause more serious complications.

[0003] Resveratrol is a polyphenolic compound found in grapes and various plants that is known for its antioxidant, anti-inflammatory, and lipid-lowering properties. Resveratrol has been shown to modulate cholesterol metabolism through various mechanisms, including activation of AMP-activated protein kinase (AMPK). AMPK activation inhibits hepatic cholesterol synthesis and promotes fatty acid oxidation, which may reduce the risk of gallstone formation. In addition, the antioxidant properties of resveratrol can alleviate oxidative stress, a factor that contributes to the development of dyslipidemia and other metabolic disorders that lead to gallstone formation. However, the role of resveratrol in regulating cholesterol metabolism in the liver itself and cholesterol gallstones is less clear.

[0004] Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that regulate the expression of genes involved in lipid metabolism, energy homeostasis, and inflammation. Three subtypes of PPARs have been identified—PPARα, PPARβ / δ, and PPARγ—each of which plays a different role in different tissues. PPARγ, in particular, is involved in lipogenesis and lipid storage and is highly expressed in adipose tissue, liver, and other organs. Activation of PPARγ has been shown to regulate inflammatory responses. Studies have shown that activation of PPARγ may reduce the production of proinflammatory cytokines and inhibit inflammatory pathways associated with gallstone development, such as NF-κB. In addition, receptors such as RAGE (receptor for advanced glycation end products) on hepatocytes have been shown to activate inflammatory pathways that lead to gallstone formation. By regulating PPAR signaling, it is possible to reduce inflammation and improve bile composition, providing a theoretical basis and potential therapeutic strategy for cholesterol gallstone disease.

[0005] In the prior art, drug treatment options for cholesterol gallstones are primarily aimed at dissolving the stones or preventing their formation. Ursodeoxycholic acid (UDCA) is a commonly used drug that works by reducing the cholesterol saturation in bile and increasing its fluidity, allowing small cholesterol stones to dissolve. However, UDCA has limited effects on larger stones, and treatment usually requires prolonged use. In addition, UDCA therapy does not address the underlying metabolic issues that lead to gallstone formation. Other drugs, such as chenodeoxycholic acid and lithocholic acid, have also been explored with mixed success rates. Surgical intervention, usually cholecystectomy, remains the standard treatment for gallstones. However, this surgery carries risks including infection, bile duct damage, and the potential need for further surgical intervention. Therefore, emerging research on natural compounds such as resveratrol and PPAR pathway modulation provides promising new directions for managing cholesterol gallstones. Addressing the underlying metabolic and inflammatory processes may not only improve treatment outcomes, but also reduce the significant healthcare burden associated with this common and painful disease. Summary of the invention

[0006] In view of the shortcomings of the prior art, the purpose of the present invention can be achieved through the following technical solutions:

[0007] Application of resveratrol in preparing medicine for preventing or treating gallstones.

[0008] Furthermore, the gallstones include cholesterol gallstones.

[0009] Application of resveratrol in preparing medicine for alleviating gallbladder damage.

[0010] Furthermore, the gallbladder injury includes the hyperplasia of the gallbladder wall lamina propria mucosa and connective tissue.

[0011] Application of resveratrol in preparing medicine for inhibiting liver inflammation.

[0012] Application of resveratrol in preparing drugs for reducing cholesterol saturation.

[0013] A method for constructing a mouse model for exploring the effect of resveratrol on gallstones, the method comprising the following steps:

[0014] S1. Specific pathogen-free healthy male mice were fed with basic feed to adapt to the environment, then weighed and recorded, and randomly divided into four groups, namely control group, model group, ursodeoxycholic acid group and resveratrol group;

[0015] S2, ursodeoxycholic acid group was fed with high-fat and high-cholesterol lithotripsy feed and gavage with ursodeoxycholic acid; resveratrol group was fed with high-fat and high-cholesterol lithotripsy feed and gavage with resveratrol; control group was fed with basal feed and gavage with sodium carboxymethylcellulose; model group was fed with high-fat and high-cholesterol lithotripsy feed and gavage with 5% sodium carboxymethylcellulose; they were grown under standard laboratory conditions and tube-fed once a day;

[0016] S3. After 5 weeks, mice were anesthetized by intraperitoneal injection of 4% chloral hydrate and euthanized by eye blood sampling; the upper serum was obtained for biochemical assays;

[0017] S4. After photographing the overall appearance of the mice, the liver, gallbladder, and bile were collected from each mouse, photographed and measured in turn, the gallbladder wall was observed and measured under a microscope, the liver was weighed, and the cholesterol crystals in the bile were analyzed.

[0018] Furthermore, in step S2, when feeding ursodeoxycholic acid, ursodeoxycholic acid needs to be dissolved in physiological saline; when feeding resveratrol, resveratrol powder needs to be dissolved in sodium carboxymethylcellulose solution.

[0019] Furthermore, in step S2, the gavage amount of ursodeoxycholic acid, resveratrol, sodium carboxymethyl cellulose and 5% sodium carboxymethyl cellulose is 100 mg / kg / d.

[0020] Further, the specific steps for analyzing bile cholesterol crystals are:

[0021] A1. One drop of bile was randomly collected from the gallbladders of mice in the control group, model group, ursodeoxycholic acid group, and resveratrol group for smear examination;

[0022] A2. Prepare pathological sections of gallbladder and liver and perform HE, Masson staining, and immunohistochemical protein blotting analysis;

[0023] A3. The collected gallbladder bile and serum are tested for cholesterol, phospholipids, and total bile acid using a biochemical analyzer.

[0024] Beneficial effects of the present invention:

[0025] 1. The present invention provides the use of resveratrol in the prevention and treatment of gallstones through PPARγ. Resveratrol regulates liver cholesterol metabolism, reduces bile cholesterol saturation and relieves liver inflammation by upregulating the expression and function of PPARγ in the liver, significantly reduces gallstone formation and relieves related symptoms such as gallbladder dilatation and cholestasis. It has the advantages of novel action targets, reliable efficacy, no toxic side effects, abundant resources, and low price.

[0026] 2. Resveratrol can be used to prepare choleretic drugs or health products, and to prepare drugs for treating cholestasis or gallstones, by upregulating the expression and function of PPARγ in the liver.

[0027] 3. This application explains the regulatory mechanism of resveratrol in gallstone formation, which prevents the formation of cholesterol gallstones in mice by increasing the expression of PPARγ and SR-BI, promoting cholesterol efflux and reducing cholesterol levels; inhibiting the production of RAGE, reducing bile cholesterol saturation and regulating the expression of proteins involved in liver cholesterol metabolism to reduce liver inflammation. This provides a theoretical basis for the development of preventive drugs for corresponding diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 Resveratrol of the present application can reduce the occurrence of gallstones. (A) Representative images of the gallbladder appearance of mice in the normal group, HF, UDCA+HF and Res+HF groups. HF: high fat diet, UDCA: ursodeoxycholic acid, Res: resveratrol. (B) Polarized light microscopy images showing bile crystals in the gallbladder of mice in each treatment group.

[0030] Figure 2 The resveratrol of the present application reduces gallbladder damage in mice. (A) Representative histological images of gallbladder sections stained with hematoxylin and eosin (H&E). (B) Quantification of gallbladder wall thickness in different experimental groups. (C) Representative Masson trichrome staining showing collagen deposition in the gallbladder wall. (D) Measurement of gallbladder volume in different experimental groups.

[0031] Figure 3 Resveratrol of the present application inhibits liver inflammation by inhibiting the production of RAGE. (A) Representative IHC staining of IL-6 in liver tissues from four experimental groups. (B) Representative IHC staining of RAGE in liver tissues from four experimental groups. (C) Quantitative analysis of IL-6 staining intensity, represented by a histogram, showing enhanced staining. (D) The histogram depicts the percentage of RAGE immunohistochemistry-positive area in liver tissue. (E) Western blot analysis of IL-6 protein levels in liver tissue samples. (F) Quantitative expression of IL-6 protein relative to the control group. (G) Western blot analysis of NF-κB and RAGE protein expression in liver tissue. (H) Quantitative expression of RAGE protein relative to the control group. (I) Quantitative expression of NF-κB protein relative to the control group.

[0032] Figure 4 Figure 2. Changes in body weight and hepatic lipid deposition in mice fed a high-fat diet for the present application. (A) Weekly weight changes in mice. (B) Total weight gain in different treatment groups during the study. (C) Representative H&E staining images of liver tissues of mice in the four experimental groups. (D) Liver weight measurements for each treatment group. (E) Serum aspartate aminotransferase (AST) levels in different groups. (F) Cholesterol content in the biliary system of mice. (G) Phospholipid levels in the bile of treated and control mice. (H) Bile acid content in the bile of mice.

[0033] Figure 5 Effects of resveratrol on protein expression in liver cholesterol metabolism for the present application (A) Representative IHC staining of PPAR-γ in liver tissues of the four experimental groups. (B) Quantification of PPAR-γ staining intensity, represented by histograms. (C and D) Western blot analysis showing the expression of PPAR (C) and SR-BI (D) proteins. (E) Relative expression levels of PPAR proteins from western blot analysis. (F) Relative expression levels of SR-BI proteins from western blot analysis. DETAILED DESCRIPTION

[0034] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Example

[0036] Thirty-two specific pathogen-free (SPF) healthy male C57BL / 6 mice, aged 6 weeks, were obtained from Nantong Trofeo Feed Technology Co., Ltd. They were fed with a basic diet for 1 week and then weighed and recorded. Then they were randomly divided into 4 groups, 8 mice in each group, namely the control group, model group (HF), ursodeoxycholic acid group (UDCA+HF) and resveratrol group (Res+HF). The ursodeoxycholic acid group (UDCA+HF) was fed with a high-fat and high-cholesterol stone-producing diet and gavaged with 100 mg / kg / d ursodeoxycholic acid; the resveratrol group (Res+HF) was fed with a high-fat and high-cholesterol stone-producing diet and gavaged with 100 mg / kg / d resveratrol; the control group was fed with a basic diet and gavaged with an equal volume of sodium carboxymethylcellulose / d; the model group (HF) was fed with a high-fat and high-cholesterol stone-producing diet and gavaged with an equal volume of 5% sodium carboxymethylcellulose / d. The animals were grown under standard laboratory conditions and gavaged once a day. Ursodeoxycholic acid was dissolved in saline, and resveratrol powder was dissolved in sodium carboxymethylcellulose solution. The mice were weighed and recorded once every evening.

[0037] After 5 weeks, mice were euthanized by intraperitoneal injection of 4% chloral hydrate (300 mg / kg body weight) for anesthesia and blood sampling was performed on the eyeball. The upper serum was obtained for subsequent biochemical determinations. After photographing the overall appearance of the mice, the liver, gallbladder, and bile were collected from each mouse, photographed and measured in turn, and the gallbladder wall was measured under a microscope. The liver was weighed.

[0038] Analysis of bile cholesterol crystals: One drop of bile was randomly collected from the gallbladders of 5 mice in each of the control group, model group, ursodeoxycholic acid group and resveratrol group for smear examination.

[0039] Pathological sections of the gallbladder and liver were prepared and subjected to HE, Masson staining, immunohistochemical protein blot analysis, etc. The collected gallbladder bile and serum were tested for cholesterol, phospholipids, total bile acid (TBA) and other indicators using a biochemical analyzer.

[0040] Experimental results analysis:

[0041] Results 1: The therapeutic effect of resveratrol on gallstones

[0042] like Figure 1 The gallbladder stones of mice were observed to be yellowish, granular, sandy, or irregularly shaped deposits, and bile was clearly visible through the gallbladder wall. Visual observation of the gallbladders of each group showed that the model group had larger and more numerous stones and significantly more turbid bile compared with the other groups. In contrast, the control group, resveratrol group, and ursodeoxycholic acid group all showed smaller and fewer stones, and clear bile ( Figure 1 Polarized light microscopy analysis of bile from each group ( Figure 1B) in the figure shows that the stone group contained a large number of typical cholesterol hydrate crystals, while the resveratrol and ursodeoxycholic acid groups showed only dispersed cholesterol crystals. The crystal size and number of these groups were significantly lower than those of the control group. These findings suggest that resveratrol effectively reverses gallstone formation and relieves symptoms of gallbladder dilatation and cholestasis.

[0043] Result 2: Resveratrol can alleviate gallbladder damage.

[0044] Histological analysis of mouse gallbladder tissue using H&E staining ( Figure 2 A) in the figure shows that the control group showed no signs of congestion or significant inflammatory cell infiltration. In contrast, the model group showed extensive inflammatory cell infiltration in the gallbladder tissue. However, both the resveratrol and ursodeoxycholic acid treatment groups showed significant improvement, with minimal edema and only a small amount of inflammatory cell infiltration.

[0045] In mice induced by a high cholesterol diet, the gallbladder wall showed hyperplasia of the lamina propria mucosa and connective tissue ( Figure 2 C), while small blood vessels and collagen fibers increased significantly. Treatment with resveratrol and ursodeoxycholic acid alleviated gallstone-induced damage and restored gallbladder wall thickness and structural integrity to levels similar to those of the control group. The model group showed significantly thicker gallbladder wall and larger gallbladder volume compared with the control and treatment groups. Notably, the gallbladder wall thickness and volume were the smallest in the resveratrol group (Table 1, Figure 2 D).

[0046] Table 1 Schematic diagram of gallbladder volume data

[0047]

[0048]

[0049] Result 3: Resveratrol inhibits liver inflammation by inhibiting the production of RAGE.

[0050] Liver inflammation plays a crucial role in the pathogenesis of gallstone formation. The receptor for advanced glycation end products (RAGE), upon binding to its ligand, activates various cell signaling pathways and subsequently upregulates the NF-κB transcription factor. This leads to increased production of inflammatory cytokines and triggers an inflammatory response. Western blot and immunohistochemistry analysis of RAGE, inflammatory cytokine IL-6, and NF-κB expression levels were performed ( Figure 3 The results showed that in the high-fat diet group treated with resveratrol, the expression levels of RAGE and inflammatory cytokines (IL-6, NF-κB, etc.) were significantly reduced. In contrast, the high-fat diet group that did not receive resveratrol showed significantly increased levels of these inflammatory mediators ( Figure 3These results suggest that resveratrol may alleviate gallstone formation by inhibiting RAGE expression, thereby inhibiting the synthesis of proinflammatory cytokines.

[0051] Result 4: Resveratrol reduces liver lipid deposition and lowers cholesterol saturation

[0052] Experimental data collected over five weeks showed that ( Figure 4 A) HF-fed C57BL / 6 mice showed significant weight gain compared with the control group. The weight gain of hyperlipidemic mice was significantly higher than that of normal mice ( Figure 4 In these groups, the body weights of HF and Res+HF mice showed significant differences. Microscopic observation of liver tissue after H&E staining showed that in the HF group ( Figure 4 In C), hepatocytes were enlarged with marked fatty degeneration, accompanied by varying degrees of inflammatory cell infiltration. In addition, signs of hepatocyte enlargement, focal necrosis, fragmented necrosis, and bile stasis were observed. Compared with the HF group, liver tissue lesions were milder in the ursodeoxycholic acid (UDCA) and resveratrol-treated groups. Mice fed a high-cholesterol diet that led to gallstone formation showed liver damage, including hepatomegaly. However, resveratrol treatment alleviated the extent of liver damage.

[0053] Liver weight measurement ( Figure 4 D) shows that both UDCA and resveratrol groups significantly reduced liver weight, which was closer to the normal level observed in the control group compared with the HF group. Notably, the ratio of liver weight to body weight was significantly reduced in the resveratrol group, indicating that the observed changes in liver weight were independent of body weight, thus excluding the potential confounding effect of body weight on the data.

[0054] Aspartate aminotransferase (AST) is a key marker of liver damage. The serum data of mice clearly showed that the AST level in the HF group was higher than that in the control and treatment groups ( Figure 4 E) in the figure further confirmed the presence of liver damage.

[0055] Bile cholesterol saturation is closely related to cholesterol metabolism. Abnormal cholesterol metabolism can disrupt the concentration of cholesterol in bile, which in turn affects bile cholesterol saturation and increases the risk of cholesterol gallstone formation. In the treatment group, the cholesterol content in the gallbladder bile of mice was significantly reduced ( Figure 4 F in Figure 1). Resveratrol was found to reduce bile cholesterol saturation, and both the UDCA+HF and Res+HF groups reduced bile phospholipid levels to values ​​similar to those of the control group ( Figure 4 G in the figure). Compared with the HF group, the bile acid levels in the UDCA+HF and Res+HF groups were significantly increased, indicating that resveratrol promoted the increase of bile acid content in the bile of mice ( Figure 4 H in.

[0056] Result 5: Resveratrol regulates liver cholesterol metabolism and alleviates gallstone formation by regulating PPAR-γ and SR-BI expression

[0057] Western blotting and immunohistochemistry were used to detect the expression level of PPAR-γ. The results showed that the expression of PPAR-γ in the resveratrol-treated group was significantly increased, while its expression in the stone group was significantly decreased ( Figure 5 A in 5 to C in 5). Many studies have shown that PPAR-γ can regulate SR-BI expression, which is a process that controls lipid and cholesterol homeostasis in hepatocytes and macrophages. Western blot analysis showed that SR-BI expression was significantly increased in the resveratrol-treated group, promoting cholesterol efflux, thereby reducing cholesterol levels and alleviating gallstone symptoms ( Figure 5 D in , E in 5).

[0058] In this application, resveratrol is used as a PPARγ modulator to upregulate the expression and function of PPARγ in the liver, thereby affecting a series of inflammatory factors, regulating liver cholesterol metabolism, reducing bile cholesterol saturation and relieving liver inflammation, significantly reducing gallstone formation and relieving related symptoms, and has the advantages of reliable efficacy and low price.

[0059] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0060] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. The use of resveratrol in the preparation of a drug for preventing or treating gallstones.

2. The use according to claim 1, characterized in that The gallstones include cholesterol gallstones.

3. Application of resveratrol in the preparation of drugs for alleviating gallbladder damage.

4. The use according to claim 3, characterized in that The gallbladder injury includes the proliferation of the gallbladder wall lamina propria mucosa and connective tissue.

5. Application of resveratrol in the preparation of drugs for inhibiting liver inflammation.

6. Use of resveratrol in the preparation of drugs for reducing cholesterol saturation.

7. A method for constructing a mouse model for exploring the effect of resveratrol on gallstones, characterized in that: The construction method comprises the following steps: S1. Specific pathogen-free healthy male mice were fed with basic feed to adapt to the environment, then weighed and recorded, and randomly divided into four groups, namely control group, model group, ursodeoxycholic acid group and resveratrol group; S2, ursodeoxycholic acid group was fed with high-fat and high-cholesterol lithotripsy feed and gavage with ursodeoxycholic acid; resveratrol group was fed with high-fat and high-cholesterol lithotripsy feed and gavage with resveratrol; control group was fed with basal feed and gavage with sodium carboxymethylcellulose; model group was fed with high-fat and high-cholesterol lithotripsy feed and gavage with 5% sodium carboxymethylcellulose; they were grown under standard laboratory conditions and tube-fed once a day; S3. After 5 weeks, mice were anesthetized by intraperitoneal injection of 4% chloral hydrate and euthanized by eye blood sampling; the upper serum was obtained for biochemical assays; S4. After photographing the overall appearance of the mice, the liver, gallbladder, and bile were collected from each mouse, photographed and measured in turn, the gallbladder wall was observed and measured under a microscope, the liver was weighed, and the cholesterol crystals in the bile were analyzed.

8. The method for exploring the effect of resveratrol on gallstones according to claim 4, characterized in that: In step S2, when feeding ursodeoxycholic acid, ursodeoxycholic acid needs to be dissolved in physiological saline; when feeding resveratrol, resveratrol powder needs to be dissolved in sodium carboxymethylcellulose solution; the feeding amount of ursodeoxycholic acid, resveratrol, sodium carboxymethylcellulose and 5% sodium carboxymethylcellulose in step S2 is 100 mg / kg / d.

9. The method for exploring the effect of resveratrol on gallstones according to claim 4, characterized in that: The specific steps for analyzing bile cholesterol crystals are: A1. One drop of bile was randomly collected from the gallbladders of mice in the control group, model group, ursodeoxycholic acid group, and resveratrol group for smear examination; A2. Prepare pathological sections of gallbladder and liver and perform HE, Masson staining, and immunohistochemical protein blotting analysis; A3. The collected gallbladder bile and serum are tested for cholesterol, phospholipids, and total bile acid using a biochemical analyzer.

10. A drug for preventing or treating gallstones, characterized in that: The medicine contains resveratrol.

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