The Protective Effects And Mechanisms Of Angelica Sinensis Polysaccharide On Liver And Pancreas Islet Injury In Type 2 Diabetic Mice

Angelica sinensis(the dry root of Angelica sinensis(Oliv.)Diels),is mainly distributed in Minxian,Yunnan and Sichuan.As a traditional Chinese herb both used in food and medicine,Angelica sinensis has been implicated in blood tonification and promotion of blood circulation.Studies have shown that Angelica sinensis polysaccharide(ASP),the major ingredient of Angelica sinensis,possesses various bioactivities,including hematopoiesis,immunomodulation and anti-oxidation.Our group has been focused on investigating the extraction and purification methods and related biological activities of Angelica sinensis polysaccharide for more than ten years.Our preliminary exploration showed that Angelica sinensis polysaccharide had beneficial effects on improving the metabolic disorders of BALB/c mice with type 2 diabetes mellitus(T2DM)and repairing accompanied liver and pancreatic islet failures.Unfortunately,further studies about these pharmacodynamic effects were rarely mentioned.In this study,Angelica sinensis polysaccharide was obtained from Angelica sinensis in Minxian by using established processes;in vivo experiments mainly focused on liver and pancreatic islets in T2DM mice induced by high-fat diet(HFD)and streptozotocin(STZ);in vitro experiments mainly focused on human hepatocellular carcinoma cell line HepG2 and mouse insulinoma pancreatic isletβcell lineβ-TC6;we aimed to investigate the protective effects and mechanisms of Angelica sinensis polysaccharide on liver and pancreatic islets injury in mice with T2DM(Figure 1).Figure 1 Topics of this subject“the protective effects and mechanisms of Angelica sinensis polysaccharide on liver and pancreatic islet injury in T2DM mice”Part Ⅰ.Angelica sinensis polysaccharide regulates glucose-lipid metabolisms in type 2 diabetes mellitus(T2DM)miceWater-boiling extraction method was used to obtain Angelica sinensis polysaccharide water solution,and then crude Angelica sinensis polysaccharide was isolated by using a series of methods including pH regulation,alcohol precipitation and drying.After repeated freeze dissolution and dialysis,Angelica sinensis polysaccharide was ultimately prepared through a gel filtration chromatograph with Sephadex-G50 and lyophilization.The sugar content of Angelica sinensis polysaccharide was 92.08%;the relative molecular weight was1.003×10~5 Da,and protein and nucleic acid were absent.This Angelica sinensis polysaccharide has the advantages of stable yield,high purity and uniform molecular weight,therefore being suitable for further investigating biological activities or medicinal values in animal or cell experiments.BALB/c mice with T2DM(fasting blood glucose(FBG)>11.1 mmol/L)were successfully established by feeding HFD for 8 weeks combined with a small dose of intraperitoneal injection of 66 mg/kg STZ for 4 times.After the treatment of Angelica sinensis polysaccharide for 4 weeks,it was obvious to find that the amount of drinking water,urine volume,and fecal excretion were decreased in T2DM mice,indicating that diabetic symptoms“more than three a little”were improved.FBG levels of T2DM mice were significantly decreased from approximately 13 mmol/L to 7.7 mmol/L after 4 weeks of Angelica sinensis polysaccharide treatment by using blood glucose meter.Results of oral glucose tolerance test showed that Angelica sinensis polysaccharide could improve the abilities of glucose absorption and utilization in T2DM mice,as well as repair destroyed glucose tolerance.The results of serum lipid profiles exhibited that Angelica sinensis polysaccharide could reduce the levels of triglyceride(TG),total cholesterol(TC)and low-density lipoprotein-cholesterol(LDL-C),as well as increase the levels of high-density lipoprotein cholesterol(HDL-C),thus significantly alleviating the dyslipidemia in T2DM mice.In this part,Angelica sinensis polysaccharide,extracted and purified from Angelica sinensis,was used for the treatment of T2DM mice induced by a combination of HFD and STZ.The results of basic physiological indexes related to glucose-lipid metabolisms indicated that Angelica sinensis polysaccharide could significantly improve the hyperglycemia and dyslipidemia syptoms in T2DM mice.Part Ⅱ.The protective effect and mechanism of Angelica sinensis polysaccharide on liver injury in T2DM miceAt the end of 4 weeks of Angelica sinensis polysaccharide administration,the liver was separated in order to calculate liver index,observe liver morphological and histological characteristics,and detect anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax expressions.Activities of serum alanine aminotransferase(ALT)and aspartate transaminase(AST)were also measured.The results showed that liver damage in T2DM mice was significantly relieved by the Angelica sinensis polysaccharide intervention for 4 weeks,which mainly characterized by that(1)liver index was decreased;(2)the color of the liver was gradually recovered;(3)the hepatocyte structure was more complete and lipid accumulation were decreased;(4)the activities of serum AST and ALT were significantly decreased;(5)the expression of pro-apoptotic protein Bax was reduced and anti-apoptotic protein Bcl-2 was increased.Indexes related to oxidative stress,including serum superoxide dismutase(SOD),liver catalase(CAT),glutathione peroxidase(GSH-PX),glutathione(GSH),reactive oxygen species(ROS)and malondialdehyde(MDA)were detected by biochemical method;serum proinflammatory factors TNF-αand IL-6 levels were measured by enzyme linked immunosorbent assay(ELISA);liver proteins IKKα,p-IκBαand Nuclear NF-κB p65 were determined by western blot;western blot was also applied to detect the expression of proteins SIRT1 and AMPK that participated in the regulation of liver oxidative stress,inflammatory response and apoptosis;to explore possible mechanisms of Angelica sinensis polysaccharide protected against liver injury in T2DM mice.The results showed that(1)Angelica sinensis polysaccharide enhanced the activities of SOD and GSH-PX and increased GSH content,and then promoted the removal of ROS and MDA,so as to alleviate oxidative stress in the liver of T2DM mice;(2)Angelica sinensis polysaccharide reduced the levels of serum TNF-αand IL-6,and then decreased the expressions of proteins IKKα、p-IκBαand Nuclear NF-κB p65,thus inhibiting the inflammatory response in the liver of T2DM mice;(3)The anti-inflammatory and anti-oxidative activities of Angelica sinensis polysaccharide may benefit from the activation of SIRT1-AMPK signaling pathway.Part Ⅲ.Angelica sinensis polysaccharide inhibits Nuclear NF-κB p65 and ROS by activating SIRT1-AMPK pathway in HepG2 cellsThe effects of Angelica sinensis polysaccharide and oleic acid(OA)on the viability of human hepatoma HepG2 cell were detected by MTT assay.The results showed that less than 800μg/mL Angelica sinensis polysaccharide treatment for 48 h didn’t affect HepG2cell viability;0.4~0.5 mmol/L OA stimulation for 24 h could significantly decrease the viability of HepG2 cell,and OA within 0.4~0.5 mmol/L treatment for 24 h could induce cell damage to varying degrees.The effect of Angelica sinensis polysaccharide(100,200and 400μg/mL)on the viability of HepG2 cell induced by 0.45 mmol/L OA was detected by MTT assay.The results showed that Angelica sinensis polysaccharide could enhance the viability of HepG2 cell.Flow cytometry and fluorescence microscopy were used to measure the effect of Angelica sinensis polysaccharide(100μg/mL)on the apoptosis of HepG2 cells induced by 0.45 mmol/L OA.The results showed that Angelica sinensis polysaccharide could obviously inhibit apoptosis in HepG2 cells and have the inhibition rate of more than 50%.These results demonstrated that Angelica sinensis polysaccharide had beneficial effects on protecting hepatocytes directly.MTT assay was used to detect the effect of glucose,EX527(SIRT1 inhibitor),compound C(AMPK inhibitor)and SIRT1 siRNA on HepG2 cell viability.The results showed that≤120 mmol/L glucose(48 h),≤160μmol/L EX527(24 h),≤2μmol/L Compound C(24 h)or≤200 nmol/L SIRT1 siRNA(24 h)had little effects on the activities of the cells.60 mmol/L glucose(48 h),10μmol/L EX527(24 h),2μmol/L Compound C(24 h)or 100 nmol/L SIRT1 siRNA(24 h)were used to inhibit the expressions of SIRT1and/or AMPK and p-AMPK proteins in HepG2 cells.The results showed that the expressions of ROS and Nuclear NF-κB p65 were significantly increased accompanied with the inhibition of SIRT1-AMPK pathway.In addition,Angelica sinensis polysaccharide were added to the above-mentioned HepG2 cells with impaired SIRT1-AMPK signaling for48 h,the expressions of SIRT1 and/or AMPK,p-AMPK were increased,and the contents of ROS and Nuclear NF-κB p65 were distinctly reduced.This validated the activative effect of Angelica sinensis polysaccharide on SIRT1-AMPK signaling pathway,and along with the activation of this pathway,both oxidative stress and inflammatory response mediated respectively by ROS and Nuclear NF-κB p65 were inhibited.The second part and this part systematically proved that Angelica sinensis polysaccharide activated liver SIRT1-AMPK signaling pathway,and then inhibited oxidative stress and inflammatory response,thus playing a protective role in the liver injury in T2DM mice(Figure 2).Part Ⅳ.The protective effects and mechanisms of Angelica sinensis polysaccharide on pancreatic islet function in T2DM MiceAt the end of the administration of Angelica sinensis polysaccharide,the pancreas in mice was isolated and HE staining of the pancreas was performed to observe the morphological and structural characteristics of the islet.Results showed that Angelica sinensis polysaccharide could relieve the damage of islet structure in T2DM mice.Western blot was used for the detection of the expressions of proteins mainly participated in intrinsic and extrinsic apoptosis pathways in pancreatic isletβcells,including Caspase protease family members(Cleaved caspase 8(p43 and p18),Caspase 9,Cleaved caspase 9(p39 and p37),Caspase 7,Cleaved caspase 7,Cleaved caspase 6,Caspase 3,Cleaved caspase 3(p19 and p17)and Cleaved PARP)and Bcl-2 protease family members(Bcl-2and Bax).The results showed that Angelica sinensis polysaccharide could significantly inhibit the activation of caspase family caesarean cascade-related proteins,decrease the expression of Bax and increase the expression of Bcl-2 in T2DM mice,indicating that Angelica sinensis polysaccharide reduced apoptosis of theβ-cells by blocking both the intrinsic and extrinsic apoptosis pathways.The level of fasting serum insulin was detected by ELISA;the content of insulin in pancreatic islet was detected by immunohistochemistry;the value of HOMA-βwas calculated by the formula.Results showed that insulin levels both in serum and pancreatic islets of T2DM mice were significantly increased after treatment with Angelica sinensis polysaccharide for 4 weeks,and the value of HOMA-βwas also increased,and it proved that Angelica sinensis polysaccharide could improve insulin production capacity of pancreatic islets in T2DM mice.Part Ⅴ.The effects and mechanisms of Angelica sinensis polysaccharide on alleviating the damage ofβ-TC6 cellsThe effects of Angelica sinensis polysaccharide and OA on the viability of mice insulinoma pancreatic isletβcell lineβ-TC6 were investigated by MTT assay.The results showed that less than 800μg/mL Angelica sinensis polysaccharide cultured for 48 h did not affect the activities ofβ-TC6 cells;0.4~0.8 mmol/L OA stimulation for 24 h resulted in a decrease in the activities ofβ-TC6 cells at different degrees.MTT assay and flow cytometry were used to detect the effects of Angelica sinensis polysaccharide(400μg/mL)treatment for 48 h on the activities and apoptosis ofβ-TC6 cells induced by 0.6 mmol/L OA.The results showed that Angelica sinensis polysaccharide could obviously prevent the viability inhibition and apoptosis of the cells,indicating the direct protective activity of Angelica sinensis polysaccharide onβcells.Western blot was applied to investigate the mechanisms of Angelica sinensis polysaccharide protected against the injury ofβ-TC6 cells induced by 0.6 mmol/L OA.Similarly,the expressions of major proteins that mediated the intrinsic and extrinsic apoptosis pathways inβ-TC6 cells were detected,including Caspase family members(Cleaved caspase 8(p43 and p18),Caspase 9,Cleaved caspase 9(p39 and p37),Caspase 7,Cleaved caspase 7,Cleaved caspase 6,Caspase 3,Cleaved caspase 3(p19and p17),and Cleaved PARP)and Bcl-2 family members(Bcl-2 and Bax).The results showed that Angelica sinensis polysaccharide inhibited the activation of Caspase family proteins stimulated by OA,increased the expression of Bcl-2 protein and decreased the expression of Bax,thus blocking both the intrinsic and extrinsic apoptosis pathways and reducing the apoptosis ofβ-TC6 cells.In addition,ELISA method was used to measure the insulin level ofβ-TC6 cells in response to 2.5 or 16.7 mmol/L glucose stimulation(GSIS test);RT-PCR and western blot were respectively used to detect gene and protein expressions of insulin.The results showed that Angelica sinensis polysaccharide could significantly increase the insulin level ofβ-TC6 cells in response to glucose stimulation,which was decreased by OA,and promote protein and gene expressions of insulin.It indicated that Angelica sinensis polysaccharide had beneficial effects on maintaining the insulin production ability ofβ-TC6 cells.The fourth part and this part systematically proved that Angelica sinensis polysaccharide inhibited the apoptosis of pancreatic isletβcells by blocking both the intrinsic and extrinsic apoptosis pathways,thus promoting the repairment of pancreatic islet structure and function in T2DM mice(Figure 3).