Application of liver sympathetic nerve activation inhibitor or liver sympathetic denervation in inhibiting abnormal liver glucose metabolism

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of a liver sympathetic nerve activation inhibitor or liver sympathetic denervation in inhibiting abnormal liver glycometabolism. Phosphorylation of sympathetic IRS-1 is removed from the liver, downstream PI3K / AKT signal channels are cascaded, PI3K and AKT are phosphorylated, and insulin signal transduction is promoted. By means of nerve and up-regulation sympathetic nerve innervation, an IRS-1 / PI3K / AKT signal channel can be activated, liver insulin sensitivity can be improved, liver gluconeogenesis is inhibited by mediating an AKT / FOXO1 channel, and liver glycogen synthesis is promoted by mediating an AKT / GSK-3beta channel. Liver sympathetic denervation regulates and controls glucose metabolism by activating IRS-1 / PI3K / AKT signal channels, and it is prompted that inhibition of sympathetic nerve activity or cutting off of sympathetic nerve fibers may be a treatment target of related metabolic diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of a liver sympathetic nerve activation inhibitor or liver sympathetic denervation in inhibiting abnormal liver glucose metabolism. Background Art

[0002] As the largest digestive organ and the center of material metabolism, the liver is deeply involved in the body's metabolism, detoxification, immunity and other processes, and plays a very important role in maintaining the body's normal physiological activities. The liver has extensive innervation. Not only are the hepatic blood vessels innervated by the autonomic nerves, but the hepatic parenchymal cells and non-parenchymal cells are also innervated by the sympathetic and parasympathetic nerves. A large number of nerve fibers that innervate the liver are distributed in the ligaments around the liver, including many sympathetic nerve fibers. During liver surgery, these ligaments are often free over a large area, which will inevitably damage the liver's nerve plexus and cause liver denervation.

[0003] The autonomic nerves of the liver regulate liver growth, immune effects, blood circulation, metabolism and other functions through the neurotransmitters released from their terminals. At present, some preliminary results have been obtained in the study of liver innervation, but the effect of liver denervation on liver physiology and pathology needs further study.

[0004] The liver has complex physiological and biochemical functions, as well as immune defense and regeneration functions. Anatomically, the liver is innervated by the sympathetic and parasympathetic nerves (vagus nerves), both of which belong to the autonomic nervous system. Sympathetic nerve fibers enter the liver along the portal vein and are mainly distributed around the portal vein, hepatic artery, and bile duct, while parasympathetic nerve fibers are mainly directly innervated by the hepatic branch of the vagus nerve a few millimeters above the cardia. Related studies have shown that these nerve fibers play an important regulatory role in the physiological and pathological processes of the liver and jointly regulate liver function.

[0005] Liver function is regulated by two modes of action: humoral and neural. It has been reported that sympathetic nerve fibers can directly act on hepatocytes, Kupffer cells, hepatic stellate cells, and hepatic sinusoidal endothelial cells, and stimulation of the hepatic sympathetic nerves can cause a variety of changes in body metabolism. For example, stimulation of the hepatic sympathetic nerves can lead to increased blood glucose and decreased hepatic blood flow. Stimulation of the hepatic sympathetic nerves can cause the release of lactate, the formation of uric acid and urea, and reduce the absorption of ammonia. In addition, stimulation of the sympathetic nerves can also cause changes in intrahepatic hemodynamics and block the hepatic sinusoids. The hepatic sympathetic nervous system can also regulate liver function, liver regeneration, and liver disease. The sympathetic nervous system regulates the expression of interleukin-6 and TGF-β by activating α1-AR on Kupffer cells, thereby promoting the formation of the hepatitis microenvironment and the development of hepatocellular carcinoma, suggesting that sympathetic denervation or α1-ARs blockade may be a new approach to treat HCC. In most models of hepatocyte injury, the sympathetic nervous system can also promote inflammation and fibrosis by acting on hepatic stellate cells. In summary, neural regulation plays an important role in regulating liver function.

[0006] Sympathetic nerve tone in the liver, intestine, pancreas, and peripheral tissues may be a key regulator of glucose production, insulin secretion, and peripheral glucose uptake. When the sympathetic nerves are overactive, excessive signaling may prevent these organs from properly regulating metabolic functions, such as the control of blood sugar and blood pressure. An increasing number of studies have also confirmed that activation of the sympathetic nervous system plays an important role in the pathogenesis of obesity, metabolic syndrome, diabetes, hypertension, and metabolic-related fatty liver disease.

[0007] Overactivation of the hepatic sympathetic nerves is associated with the development of cardiovascular risk, insulin resistance, metabolic syndrome, and obesity. Existing studies suggest that estrogen in the hypothalamus regulates peripheral and hepatic insulin sensitivity through sympathetic nerve signaling to the liver.

[0008] Surgical or chemical removal of the common hepatic artery sympathetic nerves can improve glucose tolerance and hepatic steatosis, and help improve glucose metabolism disorders. In addition, studies have shown that mice fed a high-fat diet show overactive hepatic sympathetic nerves and induce steatosis, while removal of hepatic sympathetic nerves can alleviate obesity-induced hepatic steatosis. Therefore, it is of scientific and practical significance to explore and study the specific mechanism by which the removal of hepatic sympathetic nerves regulates glucose and lipid metabolism. Summary of the invention

[0009] The present invention provides application of a liver sympathetic nerve activation inhibitor or liver sympathetic denervation in inhibiting abnormal liver glucose metabolism.

[0010] The present invention is achieved by the following technical solution: application of liver sympathetic nerve activation inhibitor or liver sympathetic denervation in inhibiting abnormal liver glucose metabolism.

[0011] Furthermore, the liver sympathetic denervation upregulates the phosphorylation of IRS-1, cascades the downstream PI3K / AKT signaling pathway, and phosphorylates PI3K and AKT, thereby promoting insulin signal transduction.

[0012] Following liver sympathetic denervation, insulin inhibits hepatic gluconeogenesis by activating the IRS-1 / PI3K / AKT pathway, followed by phosphorylation and inactivation of FOXO1.

[0013] Furthermore, AKT phosphorylation and enhanced AKT activity in the IRS-1 / PI3K / AKT pathway drive FOXO 1 inactivation, regulate the decreased expression of phosphoenolpyruvate carboxykinase PEPCK and glucose-6-phosphatase G6PC, and inhibit hepatic gluconeogenesis.

[0014] The present invention also provides that when the liver is denervated, insulin activates the IRS-1 / PI3K / AKT pathway, increases the phosphorylation of GSK-3β, and the inhibitory phosphorylation of GSK-3β leads to GYS activation, thereby increasing the glycogen synthesis of liver cells.

[0015] The present invention investigates the effect of liver sympathetic nerve removal on the regulation of glucose metabolism in rats. It was found that after the rat liver sympathetic nerves were removed by chemical or phenolic methods, the glucose metabolism of rats was impaired, blood sugar decreased, and glycogen increased. Through transcriptome sequencing, differentially expressed genes (DEGs) and possible signal pathways were screened to further explore the molecular mechanism of abnormal liver glucose metabolism in vivo and in vitro, and it was found that the removal of liver sympathetic nerves can enhance insulin sensitivity through the IRS-1 / PI3K / AKT pathway. In addition, the downregulation of PEPCK and G6PC mediated by IRS-1 / PI3K / AKT / FOXO 1 reduced gluconeogenesis in the liver, while the activation of GYS mediated by IRS-1 / PI3K / AKT / GSK-3β increased glycogen synthesis in hepatocytes. Inhibiting sympathetic nerve activity or cutting off sympathetic nerve fibers may be potential targets for the treatment of related metabolic diseases, which deserves further exploration and research in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Chemical denervation disrupts glucose homeostasis in the liver; Figure: A: tyrosine hydroxylase content; B: NE content; C, D: serum ALT and AST enzyme activities; E: fasting blood glucose level; F: liver tissue glycogen content; G: representative images of liver tissue sections stained with H&E and PAS; magnification 100 times; * P <0.05, ** P <0.01, *** P <0.001, **** P<0.0001. (n = 6; mean ± SD; scale bar = 100 μm); Figure 2 Phenol removes liver sympathetic nerve innervation and disturbs glucose homeostasis in the liver; In the figure: A: tyrosine hydroxylase content; B: NE content; C, D: serum ALT and AST enzyme activities; E: fasting blood glucose level; F: liver tissue glycogen content; G: representative images of liver tissue sections stained with H&E and PAS; magnification is 100 times; * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. (n = 6; mean ± SD; scale bar = 100 μm); Figure 3 The whole genome expression profile and summary of DEGs; In the figure: A: Volcano plot of 6-OHDA group and saline group; Comparison of deg differences between groups (Fold change ≥ 2.0, P < 0.05). Color indicates the level of transcriptional change (red: up-regulated; green: down-regulated); B: Volcano plot of denervation group and sham operation group. Comparison of deg differences between groups (Fold change ≥ 2.0, P < 0.05). Color indicates the level of transcriptional change (red: up-regulated; green: down-regulated). C: Cluster diagram showing the overall differences in transcription in liver tissues of rats between 6-OHDA and saline groups, as well as hepatic denervation and sham operation groups. D: Venn diagram showing deg overlap. E: Verification of mRNA expression of differentially expressed genes between 6-OHDA group and saline group. F: Verification of mRNA expression of differentially expressed genes between denervation group and sham operation group. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. (n = 6; mean ± SD); Figure 4 GO and KEGG enrichment analysis of shared DEGs; In the figure: A: GO enrichment analysis of shared DEGs; B: KEGG enrichment analysis of shared DEGs; Figure 5Sympathetic denervation affects glucose homeostasis in HepG2 cells by upregulating phosphorylation of the IRS-1 / PI3K / AKT pathway; Figure: A: Effect of PE on HepG2 cell viability; B: Glucose content in HepG2 cells: cells were treated with 0μM~100μM PE for 48h; C: Glycogen content in HepG2 cells; D: Western blotting analysis was performed using corresponding antibodies, with GAPDH as an endogenous control. "p" represents phosphorylated protein; E: Relative quantitative analysis of phosphorylated or total proteins of IRS-1, PI3K and AKT, *P<0.05, **P<0.01. (n = 6; mean ± SD); Figure 6 Effects of sympathetic denervation on key enzymes of gluconeogenesis and glycogen production in HepG2 cells; Figure: A, C: Western blotting to detect proteins related to gluconeogenesis and glycogen production in HepG2 cells. GAPDH was used as an endogenous control. "p" represents phosphorylated protein; B, D: Relative quantitative analysis of each group of proteins. *P<0.05, **P<0.01. (n = 6; mean ± SD); Figure 7 Sympathetic denervation up-regulates the phosphorylation of the IRS-1 / PI3K / AKT pathway in vivo; In the figure: A, C: Western blotting method was used to detect the proteins of the corresponding antibodies in the liver tissues of each group. GAPDH was used as the endogenous control. "p" represents phosphorylated protein; B, D: Relative quantitative analysis of proteins in each group. *P<0.05, **P<0.01. (n = 6; mean ±SD); Figure 8 Effects of sympathetic denervation on gluconeogenesis and key enzymes of glucose production in vivo; Figure: A, C: Western blotting analysis was performed and corresponding antibodies were used to detect gluconeogenesis-related proteins in liver tissues of each group. GAPDH was used as an endogenous control. "p" represents phosphorylated protein; B, D: Relative quantitative analysis of proteins in each group; E, G: Immunoblotting was used to detect proteins related to glucose production in liver tissues of each group. GAPDH was used as an endogenous control. "p" represents phosphorylated protein; F, H: Relative quantitative analysis of proteins in each group. *P<0.05, **P<0.01. (n = 6; mean ± SD); Fig. 9 This is the signaling mechanism by which sympathetic denervation of the liver leads to abnormal liver glucose metabolism. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0018] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0019] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0020] The experimental methods in the following examples are all conventional methods unless otherwise specified. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0021] The reagents used in the following examples are as follows: 6-hydroxydopamine (6-OHDA): 200 μg, 162957, Sigma-Aldrich, Shanghai, China; phenol: P100769, Aladdin, Shanghai, China; hematoxylin and eosin (H&E): G1120, Solarbio, Beijing, China; Trizol reagent: ET111-01, Tansgen, Beijing, China; high glucose-DMEM medium: Thermo Fisher Scientific, Suzhou, China; penicillin / streptomycin solution: Solarbio, Beijing, China; 10% fetal bovine serum: Procell, Wuhan, China; RIPA lysis buffer: PC101, Epizyme Biotech, Shanghai, China; protease inhibitor: AR1178, BOSTER, Wuhan, China; secondary antibody: bs-0295G-HRP, BIOSS, Beijing, China; enhanced chemiluminescence reagent: BMU102-CN, Abbkine, Wuhan, China.

[0022] Equipment used in the following examples: Microplate reader: Spectra Max 190, Molecular Devices, USA; Aspartate aminotransferase (AST) detection kit (C010-2-1), Alanine aminotransferase (ALT) detection kit (C009-2-1): Nanjing Jiancheng Company; Glycogen quantification kit: A043-1-1, Nanjing Jiancheng, Jiangsu; PA53 biological microscope: Motic, Fujian, China; PAS staining kit: G1281, Solarbio, Beijing, China; PrimeScript with gDNA Eraser TM RT kit: RR047A, Takara, Beijing, China; TB Green® Premix Ex Taq TM II: RR820A, Takara, Beijing, China; QuantStudio 3 PCR detection system: Thermo Fisher Scientific; HepG2 cells were purchased from the National Authentication Cell Culture Bank, Shanghai, China; Cell counting kit-8: BA00208, Bioss, Beijing, China; microplate reader: Spectra Max 190, Molecular Devices, USA; glucose detection kit: A154-1-1, Jiancheng, Nanjing, Jiangsu, China; glycogen determination kit: A043-1-1, Jiancheng, Nanjing, Jiangsu, China; BCA protein determination kit: ZJ102, Epizyme Biotech, Shanghai, China.

[0023] 1. Experimental Methods 1. Animal model and experimental design: Male and female Sprague-Dawley rats aged 6-8 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. These rats were provided with free access to food and were subjected to a 12-h light-dark cycle. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Shanxi Medical University and conformed to the standards of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

[0024] The following two methods were used to construct the animal model: Rats were anesthetized by isoflurane inhalation, fixed on a rat surgical board, and locally disinfected.

[0025] (1) 6-hydroxydopamine (6-OHDA) is a potent catecholamine neurotoxin that can selectively destroy sympathetic nerve endings, causing severe degeneration or even complete disappearance of sympathetic ganglia and postganglionic sympathetic neurons. When applied peripherally, it cannot cross the blood-brain barrier and is currently a recognized chemical sympathectomist.

[0026] SD rats were randomly divided into 6-OHDA group or saline group (n = 30). In the 6-OHDA-based hepatic sympathectomy experiment, a 1 cm longitudinal incision was made in the middle of the abdomen, the small intestine was gently squeezed out with forceps, the mesenteric vessels were found, and 2 ml of 6-OHDA was injected into the mesenteric vein with an insulin needle. The saline group was given 0.9% saline in the same way. The wound was pressed with a cotton swab for about 3-5 minutes, and the intestine was repositioned after bleeding. The abdominal muscle layer and skin layer were sutured, and the rats were killed at 3, 7, 14, 28, and 60 days after surgery for further analysis, and liver and serum samples were collected.

[0027] (2) Phenol is highly corrosive to the skin and mucous membranes and has a direct damaging effect on various cells. Studies have shown that the use of phenol solution can induce non-selective neurodegeneration, thereby ensuring that the sympathetic nerves are completely blocked without damaging blood vessels. In the phenol-based liver sympathetic denervation experiment, SD rats were randomly divided into a denervation group or a sham operation group (n=30). The abdominal cavity was opened along the midline incision, and the surrounding tissues such as the hepatic artery, portal vein, and bile duct were bluntly dissected. A sterile cotton swab was dipped in phenol solution (90% phenol ethanol) and then carefully applied to the surface of the hepatic artery and portal vein bundle. The abdominal cavity was flushed with 0.9% saline to remove excess phenol and reduce the formation of adhesions. The sham operation group was given 0.9% saline instead of phenol solution in the same way. The wound was sutured, and the rats were killed 3, 7, 14, 28, and 60 days after surgery to collect liver and serum samples for further analysis.

[0028] 3. Enzyme-linked immunosorbent assay (ELISA): The liver tissue was washed with PBS buffer, weighed and added with the corresponding volume of PBS buffer containing protease inhibitors, and fully ground on ice. In order to further break up the tissue cells, the tissue homogenate was treated by ultrasonic treatment. After centrifugation (3000×g, 20 min), the absorbance at 450 nm was measured using a microplate reader according to the instructions, and each measurement was performed three times.

[0029] 4. Biochemical analysis: The blood samples were centrifuged and the supernatant was collected for liver function test. The aspartate aminotransferase (AST) detection kit (C010-2-1) and the alanine aminotransferase (ALT) detection kit (C009-2-1) were used and the detection was performed using a microplate reader according to the instructions.

[0030] 5. Fasting blood glucose and glycogen quantification: All rats were allowed free access to water. After a 12-hour fasting period, tail vein blood samples were collected, and fasting blood glucose levels were assessed using blood glucose test strips and a blood glucose meter. The fasting blood glucose levels of rats in each group were monitored at 0, 3, 7, 14, 28, and 60 days after surgery.

[0031] Glycogen quantification kit was used to detect glycogen levels: after weighing the liver tissue, the corresponding amount of alkaline solution was added to the test tube in a ratio of sample weight (mg): alkaline solution volume (ul) = 1:3, and then heated in a boiling water bath for 20 minutes. After cooling, the glycogen detection solution was prepared by adding an appropriate amount of double distilled water for subsequent sample addition and color development.

[0032] 6. Histopathology: Fresh liver tissue was fixed in 4% paraformaldehyde for 24 hours, dehydrated, wax-impregnated, embedded, and sliced ​​to prepare paraffin sections. 4 μm paraffin sections were dewaxed and stained with hematoxylin and eosin (H&E). After staining, the sections were dehydrated, sealed, and examined using a PA53 biological microscope. Image J software was used for image acquisition and analysis.

[0033] 7. PAS staining: PAS staining of intracellular glycogen deposition was performed using a PAS staining kit: liver paraffin sections were dewaxed, hydrated, and oxidized with hydrochloric acid at room temperature for 8 minutes. Then, the sections were washed three times with distilled water, shaken to remove excess water, and stained with Schiff staining solution at room temperature in a dark place for 10-20 minutes. After rinsing with tap water for 10 minutes, the sections were stained with hematoxylin staining solution for 1-2 minutes and differentiated with acid differentiation solution for 2-5 seconds. Finally, the sections were rinsed with tap water for 10-15 minutes, dehydrated in gradient alcohol, and sealed with neutral gum. Purple or purple-red glycogen staining was observed under a PA53 biological microscope.

[0034] 8. RNA sequencing and mRNA expression analysis of differentially expressed genes: In the early stage of the experiment, a rat sympathetic denervation model was constructed, and liver tissues from each group were collected and stored in liquid nitrogen. RNA extraction, cDNA library construction, quality control and sequencing were all completed by Shanghai Sangong Biotechnology Co., Ltd. The Illumina Xten platform was used for sequencing. After quality control, the original sequencing data was aligned with the reference genome sequence, the reads of the gene were counted, and the expression of the gene was calculated. DESeq2 was used to analyze differentially expressed genes (DEGs), and the results were visualized. The screening conditions were set to q value <0.05 and difference fold |Fold Change|>2 to obtain up-regulated and down-regulated differentially expressed genes. According to the results of the differential analysis, uniform maps and heat maps were drawn, and cluster analysis was performed. Gene ontology (GO) analysis and KEGG enrichment analysis were performed.

[0035] 9. Real-time quantitative PCR (qRT-PCR): Total RNA was extracted from liver tissue and cells using Trizol reagent according to the manufacturer's protocol and PCR was performed using PrimeScript with gDNA Eraser. TMRT kit to obtain complementary DNA (cDNA) from total RNA. Next, TB Green® Premix Ex Taq TM II for real-time fluorescence quantitative PCR (qRT-PCR). PCR reactions were analyzed using the QuantStudio 3 PCR detection system. The expression level of the target gene was normalized to that of the GAPDH gene and 2 -ΔΔCt Methods The relative expression of mRNA was calculated. All reactions were performed in triplicate, and all primer sequences were designed and synthesized by Sangon Biotech (Shanghai, China) and are shown in Table 1.

[0036] Table 1 RT-qPCR specific primer sequences 10. Cell culture: HepG2 cells were purchased from the National Authentication Cell Culture Bank (Shanghai, China). HepG2 cells were routinely cultured in high glucose-DMEM medium, supplemented with penicillin / streptomycin solution and 10% fetal bovine serum, and cultured in a humidified incubator at 37°C and 5% CO2 atmosphere.

[0037] 11. CCK-8: Cell counting kit-8 was used to detect cell viability. After HepG2 cells were treated with norepinephrine 0-40 μg / mL, 3×10 3 Cells were inoculated into each well of a 96-well plate, with 5 replicate wells set for each concentration. After 24 h of culture, 10 μl of CCK8 solution was added to each well, incubated for 2 h, and then the absorbance of each well at 450 nm was measured using an ELISA reader. The experiment was repeated three times, and all experimental steps were performed according to the instructions of the kit.

[0038] 12. Cell glucose and glycogen quantification: According to the instructions of the glucose detection kit, the glucose oxidase method was used to detect the glucose content in the supernatant of each group of cell culture medium. According to the instructions of the glycogen determination kit, the colorimetric method was used to detect the glycogen content of each group: after the cell precipitate was collected and washed, 225μl of alkaline solution was added to each tube and boiled in a water bath for 20min. Then 225μl of double distilled water was added and cooled with tap water to prepare a glycogen detection solution. Subsequent operations were carried out according to the instructions, and the absorbance of each group of cell samples was detected at a wavelength of 620nm.

[0039] 13. Immunoblotting analysis: Tissues or cells were collected, RIPA lysis buffer and protease inhibitors were added to make a mixed homogenate, and then incubated on ice for 30 minutes. The homogenized samples were centrifuged at 10,000 g for 15 minutes at 4°C. The supernatant was collected for further analysis. The protein concentration of the supernatant was determined using a BCA protein assay kit and modified to the same level with lysis buffer. The protein buffer was then mixed evenly with the sample protein in a 1:4 ratio and heated at 100°C for 10 minutes to denature the protein. Subsequently, equal amounts of protein were separated by 10% SDS-PAGE and transferred to a PVDF membrane. The protein-containing PVDF membrane was blocked with 5% skim milk in tris-buffered saline containing 0.1% Tween-20 (TBST) at room temperature for 2 hours, incubated with the designated primary antibody (appropriate dilution) at 4°C overnight, and then incubated with the secondary antibody at 4°C for 2 hours. The protein bands were visualized with an enhanced chemiluminescence reagent and quantified using Image J software. The signal intensity of each band was normalized to the signal intensity of the GAPDH band loaded on the same membrane. All primary antibodies used in the present invention are shown in Table 2.

[0040] Table 2 Antibodies used in the present invention 14. Statistical analysis: All statistical calculations were performed using a statistical software program (GraphPad Prism 9.0 software, USA). Data are presented as mean ± standard deviation (mean ± SD). The independent sample T test was used for comparison between the two groups. All statistical comparisons were considered statistically significant when p < 0.05.

[0041] 2. Experimental Results 1. Chemical denervation disrupts glucose homeostasis in the liver: TH is the rate-limiting enzyme for catecholamine synthesis in sympathetic postganglionic nerve endings (a sign of sympathetic intrinsic innervation). The results showed that compared with the saline group, the expression of TH in the rat liver was significantly reduced at 3d, 7d, 14d, 28d and 60d after 6-OHDA administration ( Figure 1 This was further verified by measuring the NE content in liver tissue (A). Figure 1 B) These results indicate that sympathetic nerves in the rat liver have been destroyed.

[0042] ALT and AST levels are commonly used to evaluate liver function. Next, the serum ALT and AST levels of the two groups of rats were tested at different time points. The results showed that the serum ALT and AST levels of the rats in the 6-OHDA group were significantly increased compared with those in the saline group ( Figure 1 C, D), suggesting that hepatic sympathetic denervation affects liver function to a certain extent.

[0043] To investigate whether hepatic sympathectomy disrupts glucose homeostasis in rats, fasting blood glucose and liver tissue glycogen content were measured in the two groups of rats at different time points. Figure 1 As shown in E and F, a decrease in fasting blood glucose and an increase in liver tissue glycogen content were observed at 5 periods (3d, 7d, 14d, 28d and 60d) after 6-OHDA injection. Figure 1 As shown in E, 14 days after hepatic sympathetic nerve removal, the glucose content in liver tissue was significantly reduced, indicating that gluconeogenesis was significantly inhibited. Figure 1 As shown in F, a significant increase in the glycogen content of liver tissue was observed 14 days after hepatic sympathetic denervation, while the increase in glycogen content in liver tissue treated at other time points was less significant. Figure 1 G, which was further confirmed by HE and PAS staining. The results showed that glucose homeostasis in rat hepatocytes was most severely disturbed 14 days after hepatic sympathetic denervation.

[0044] 2. Phenol-based hepatic sympathetic denervation interferes with glucose homeostasis in the liver: Nonselective hepatic sympathectomy was performed, and similar results were obtained as with 6-OHDA chemical sympathectomy. Figure 2 As shown in A, the content of TH in the liver tissue of the denervated rats was significantly reduced compared with that of the sham-operated rats, which confirmed the effectiveness of this denervation method. Figure 2 The method was further validated by measuring the NE content in liver tissue as shown in B. In addition, it was found that the content of TH and NE in liver tissue decreased to the lowest level on the 14th day after phenol was removed from the liver sympathetic nerves (as shown in Figure 2 (as shown in A and B).

[0045] Then the serum ALT and AST levels of the two groups of mice were measured. Figure 2 As shown in C and D, the results showed that the serum ALT and AST levels of rats in the denervated group were significantly increased compared with those in the sham group. These findings suggest that hepatic sympathetic denervation using phenol also affects liver function.

[0046] Next, to investigate whether phenol sympathectomy disrupted intracellular glucose homeostasis in rats, blood glucose levels and glycogen content in liver tissue were measured in fasting rats. Figure 2 As shown in E and F, compared with the sham operation group, the fasting blood glucose level of the denervated rats was reduced and the glycogen content in the liver tissue was increased at five time points: 3d, 7d, 14d, 28d, and 60d after hepatic sympathetic nerve resection. Figure 2G shows that at 14 days after sympathectomy, glucose levels were significantly reduced and glycogen levels were increased in the denervated group, whereas the increase in glycogen levels was not significant in livers treated at other time points, as further visualized by HE and PAS staining. These significant findings are consistent with chemical sympathetic denervation (6-OHDA). These results suggest that glucose homeostasis is most severely perturbed 14 days after hepatic sympathectomy.

[0047] Therefore, in subsequent studies, an animal model 14 days after hepatic sympathetic nerve transection was used to further explore the molecular mechanism of abnormal hepatic glucose metabolism.

[0048] 2. Whole-genome expression profile and differentially expressed gene analysis: To further explore whether the molecular mechanisms of abnormal hepatic glucose metabolism caused by chemical denervation of hepatic sympathetic nerves by 6-OHDA or non-selective denervation of sympathetic nerves by phenol are consistent, transcriptome analysis was used to explore the key genes hidden in the cross-action and the potential mechanisms. 433 differentially expressed genes (DEGs) were screened from the saline group (n = 3) and the 6-OHDA group (n = 3), including 253 upregulated genes and 180 downregulated genes (see Figure 3A). In addition, a total of 957 DEGs were identified from the sham operation group (n = 3) and the denervation group (n = 3), covering 842 upregulated genes and 115 downregulated genes (see Figure 3B). Subsequently, a cluster analysis was carried out on the DEGs of each group, and the heat map results are shown in Figure 3 C. The Veen plot can be used to present the distribution of DEGs between groups and the intersection of DEGs. Then, the intersection of the selected DEGs was obtained, and a total of 158 common differentially expressed genes were obtained, of which 119 were co-upregulated genes and 39 were co-downregulated genes (see Figure 3D).

[0049] To verify the results of transcriptome sequencing, genes with significant differences such as Gck, Mapk10, Ppp1r3c, Fasn, Srebf1, Osmr, Thbs1 and Cdkn1a were selected for RT-qPCR verification. The results showed that compared with the saline group, the expression of Gck and other genes in the liver tissue of rats in the 6-OHDA group was significantly increased (P<0.05) (see Figure 3E). Similarly, compared with the sham operation group, the expression of genes such as Gck in the denervation group in the liver tissue of rats was significantly increased (P<0.05), which was consistent with the results of transcriptome sequencing (see Figure 3F).

[0050] 3. GO and KEGG enrichment analysis of differentially expressed genes: GO and KEGG enrichment analysis were performed to reveal the potential functions of these DEGs. For GO enrichment analysis, the common DEGs mainly involved phylogeny, multicellular biological development, anatomical structure development, developmental process, positive regulation of cellular processes, ion binding, and catalytic activity (see Figure 4A). For KEGG pathway enrichment analysis, the most significant pathways included circadian rhythm, mineral absorption, FcγR-mediated phagocytosis, phagosome, insulin signaling pathway, phospholipase D signaling pathway, calcium signaling pathway, PI3K / AKT signaling pathway, etc. (see Figure 4B). Among them, the insulin signaling pathway is related to glucose metabolism.

[0051] 4. Sympathetic denervation affects glucose homeostasis of HepG2 cells by upregulating phosphorylation of the IRS-1 / PI3K / AKT pathway: Phenylephrine (PE) is a selective α1-adrenergic receptor agonist. HepG2 cells with PE added were used to simulate HepG2 cells with sympathetic nerve innervation under normal physiological conditions as the control group, while HepG2 cells without PE added were used as the denervation group. CCK-8 cell proliferation assay was performed on HepG2 cells under different concentrations of PE to evaluate cell survival.

[0052] The results are as follows Figure 5 As shown in A, the results show that the survival rate of HepG2 cells exposed to 20μM PE was significantly reduced compared to 0μM. CCK8 results showed that the survival rate of HepG2 cells was 83.3% after treatment with 20μM PE. Therefore, within the range of 0-20μM, more than 80% of HepG2 cells remained viable after treatment.

[0053] To investigate whether sympathetic denervation disturbed cellular glucose homeostasis, the levels of glucose and glycogen in the two groups of cells were measured. Figure 5 As shown in B and C, in the denervated group, a decrease in intracellular glucose content and an increase in glycogen content were observed.

[0054] To investigate whether sympathetic denervation induces glucose metabolism disorders through the insulin signaling pathway, we first explored the effect of denervation on IRS-1, a key component of the insulin signaling pathway at the post-receptor level, and its downstream genes. Figure 5 As shown in D and E, in HepG2 cells of the denervation group, the phosphorylation of IRS-1, PI3K, and AKT was significantly enhanced, but the total levels of IRS-1, PI3K, and AKT were not different. These data indicate that denervation can promote insulin signal transduction by upregulating the phosphorylation of IRS-1, PI3K, and AKT.

[0055] 5. Effects of sympathetic denervation on key enzymes of gluconeogenesis and glycogen production in HepG2 cells: It is well known that insulin usually downregulates blood sugar in part by inhibiting gluconeogenesis in the liver and promoting glycogen synthesis. Given the decrease in glucose content and increase in glycogen content in HepG2 cells in the denervation group, the effect of denervation on the expression of key proteins regulating gluconeogenesis and glycogen synthesis was examined next. Figure 6 As shown in A and B, denervation increased FOXO1 phosphorylation, but decreased total FOXO1 protein. In addition, PEPCK protein decreased, and G6PC also decreased, indicating that the expression of these two genes involved in gluconeogenesis was downregulated. Figure 6 As shown in C and D, the GCK protein level was increased in the denervation group. Furthermore, the phosphorylation of GSK-3β increased, while the phosphorylation of GYS decreased in HepG2 cells in the denervation group. Based on all the above results, we speculate that denervation impairs glucose homeostasis by disrupting the insulin pathway, thereby inhibiting gluconeogenesis and enhancing glycogen synthesis in HepG2 cells.

[0056] 6. Sympathetic denervation upregulates phosphorylation of the IRS-1 / PI3K / AKT pathway in vivo: The effects of chemical denervation and phenol hepatic sympathetic denervation on the expression of IRS-1, a key component of the insulin signaling pathway in liver tissue, and its downstream genes were studied. Figure 7 As shown in A and B, the phosphorylation levels of IRS-1, PI3K, and AKT in the 6-OHDA group were significantly increased compared with the saline group, while the total levels of IRS-1, PI3K, and AKT were not different. Similar results were also observed in the liver tissue of the phenol denervation group (e.g. Figure 7 These data suggest that denervation promotes insulin signaling by upregulating phosphorylation of IRS-1, PI3K, and AKT.

[0057] 7. Effects of sympathetic denervation on key enzymes of gluconeogenesis and glycogen synthesis in vivo: Next, we studied the expression of key enzymes regulating gluconeogenesis and glycogen synthesis in the liver tissue of the two rat denervation models. Figure 8 AD showed that denervation increased the phosphorylation of FOXO1 in liver tissue, but the total FOXO1 protein level was reduced. In addition, the PEPCK protein level was reduced, which was paralleled by the decrease in G6PC, indicating that the expression of these two gluconeogenic genes was downregulated. Figure 8 As shown in EH, the expression level of GCK protein in liver tissue increased significantly after denervation. In addition, the phosphorylation of GSK-3β in liver tissue increased, while the phosphorylation of GYS decreased. Given these results, it is speculated that in vivo, hepatic sympathetic denervation disrupts glucose homeostasis by interfering with the insulin pathway, thereby inhibiting gluconeogenesis and promoting glycogen synthesis.

[0058] The results of the study confirmed that after hepatic sympathetic denervation, blood glucose homeostasis in rats was disturbed, and a decrease in fasting blood glucose and an increase in glycogen content in liver tissue were observed at different time points. This result indicates that hepatic sympathetic denervation can indeed effectively reduce blood glucose levels in vivo, which deserves further discussion and research.

[0059] The liver is essential for maintaining normal blood sugar levels. Hepatic glucose production is mainly regulated by insulin and glucagon. The action of insulin involves a series of signaling cascades that are triggered by insulin binding to the insulin receptor (IR), leading to receptor autophosphorylation and activation of receptor tyrosine kinase. IRS-1 tyrosine phosphorylation is a necessary step for insulin to stimulate glucose transport. The IRS-1 / PI3K / AKT signaling pathway is a classic signaling pathway for insulin to regulate blood sugar balance. Activation of this pathway can promote insulin signaling and increase insulin sensitivity.

[0060] Once insulin binds to and activates its receptor, it promotes the phosphorylation of IRS-1, thereby activating PI3K and Akt. IRS-1 is an important factor in the cascade effect after insulin activates the receptor. It is an important substance that regulates the insulin signaling pathway and is closely related to cell growth and metabolism. Once IRS-1 is activated, a cascade reaction occurs in the downstream PI3K / AKT signaling pathway. Activated PI3K is involved in regulating cell proliferation, differentiation, glucose transport and other functions. As the main downstream signal transducer of the PI3K signaling pathway, AKT also plays an important role in lowering blood sugar in the insulin conduction pathway after activation.

[0061] To further explore the molecular mechanism of sympathetic nerve removal in liver glucose metabolism, transcriptome analysis was used to screen the differentially expressed genes (DEGs) and their intersections between the groups. KEGG pathway enrichment analysis was performed on 158 common DEGs, and the results showed that there were significant differences between the insulin signaling pathway and the PI3K / AKT signaling pathway. Therefore, the effect of sympathetic nerve removal on the IRS-1 / PI3K / AKT signaling pathway was studied in vivo and in vitro. The results showed that both in vivo and in vitro, the removal of sympathetic nerves would lead to an increase in the phosphorylation level of IRS-1, thereby cascading the downstream PI3K / AKT signaling pathway.

[0062] To further clarify whether the molecular mechanism of decreased gluconeogenesis and increased glycogen content in these studies is related to the IRS-1 / PI3K / AKT signaling pathway, key enzymes involved in gluconeogenesis and glycogen synthesis were evaluated. AKT-driven FOXO 1 activation in the liver controls hepatic gluconeogenesis by regulating the activities of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6PC). Therefore, these two genes are important target genes of FOXO 1 and are also the rate-limiting steps in catalyzing gluconeogenesis. Insulin inhibits hepatic gluconeogenesis by activating the IRS-1 / PI3K / AKT pathway, followed by phosphorylation and inactivation of FOXO 1.

[0063] In the present invention, the inactivation of FOXO 1 and the enhancement of AKT activity mediated by denervation lead to the decrease of PEPCK and G6PC expression, thereby inhibiting hepatic gluconeogenesis. This is similar to the signaling pathway by which insulin inhibits hepatic gluconeogenesis. GCK and GYS are key enzymes in the first and last steps of glycogen synthesis, and are also important downstream targets of AKT. In the present invention, it was found that consistent with the phosphorylation activity of AKT, the protein level of GCK was also significantly increased. GYS is the rate-limiting enzyme for insulin-mediated glycogen synthesis. GSK-3β is believed to mediate the occurrence of insulin resistance mainly by inhibiting glycogen synthesis. Insulin activates IRS-1 / PI3K / AKT signaling, followed by an increase in the phosphorylation level of GSK-3β, and the inhibitory phosphorylation of GSK-3β leads to the activation of GYS, thereby increasing glycogen synthesis. This is similar to the signaling pathway by which insulin promotes glycogen synthesis in the liver. Combining these data, the signaling mechanism by which liver sympathetic denervation leads to abnormal liver glucose metabolism is obtained, such as Fig. 9 As shown, it is hypothesized that hepatic denervation inhibits hepatic gluconeogenesis through IRS-1 / PI3K / AKT / fox01-mediated downregulation of PEPCK and G6PC, and promotes hepatic glycogen synthesis in hepatocytes by activating GCK and GYS.

[0064] The main new discovery of the present invention is that the removal of liver sympathetic nerves can enhance insulin sensitivity through the IRS-1 / PI3K / AKT pathway. In addition, the downregulation of PEPCK and G6PC mediated by IRS-1 / PI3K / AKT / FOXO 1 reduced gluconeogenesis in the liver, while GYS activation mediated by IRS-1 / PI3K / AKT / GSK-3β increased glycogen synthesis in hepatocytes. Inhibiting sympathetic nerve activity or cutting off sympathetic nerve fibers may be a potential target for the treatment of related metabolic diseases, which deserves further exploration and research in the future.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of hepatic sympathetic nerve activation inhibitors or hepatic sympathetic denervation in inhibiting abnormal hepatic glucose metabolism.

2. The use according to claim 1, characterized in that: The liver denervates the sympathetic nerves, upregulates the phosphorylation of IRS-1, cascades the downstream PI3K / AKT signaling pathway, phosphorylates PI3K and AKT, and promotes insulin signal transduction.

3. The use according to claim 2, characterized in that: Following liver sympathetic denervation, insulin inhibits hepatic gluconeogenesis by activating the IRS-1 / PI3K / AKT pathway, followed by phosphorylation and inactivation of FOXO1.

4. The use according to claim 3, characterized in that: AKT phosphorylation and enhanced AKT activity in the IRS-1 / PI3K / AKT pathway drive FOXO 1 inactivation, regulate the expression of phosphoenolpyruvate carboxykinase PEPCK and glucose-6-phosphatase G6PC to decrease, and inhibit hepatic gluconeogenesis.

5. The use according to claim 2, characterized in that: The liver is denervated, and insulin activates the IRS-1 / PI3K / AKT pathway, which increases the phosphorylation of GSK-3β. The inhibitory phosphorylation of GSK-3β leads to the activation of GYS, thereby increasing the glycogen synthesis of hepatocytes.

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