Effects Of Jiangtangshangshu Granule On Glucose And Lipid Metabolism And Endoplasmic Reticulum Stress In Liver Of T2DM Mice

ObjectiveIn this study, diabetes KK-Ay mice models were constructed through high-fat diet, effects of Jiang Tang Xiao Ke Granule (JTXKG) on serum and hepatic glucolipid metabolism, relative targets in insulin signaling pathway were observed and the possible underlying mechanism were explored. To observe effects of JTXKG on hepatic oxidative stress and endoplasmic reticulum stress, so to investigate the effect of JTXKG and the underlying mechanism, and to provide experimental proofs for treating diabetic liver injury with TCM methods.Methods8-week-old male KK-Ay mice were fed with high-fat diet for 4 weeks, 45 mice, whose fasting blood glucose were not less than 13.9 mmol/L, were randomly divided into 5 group (control, high dose JTXKG, medium dose JTXKG, lose dose JTXKG and pioglitazone). All mice were administered for next 10 weeks.10 C57BL/6J mice were taken as normal control group and were given standard diet. Food intake, body weight and fasting blood glucose were recorded weekly. At the 4 and 10 week, glucose tolerance were tested. All mice were sacrificed at week 10. After that, liver weight were measured for calculating of liver/body weight, serum lipids, FINS, HbAlc and free fatty acid levels were detected, serum ALT、AST、γ-GT and MDA, SOD were measured for assessing liver function and oxidative stress levels in serum, hepatic lipids and MDA level and SOD, GSH activities were detected after hepatic homogenization.For general morphological observation, hepatic tissue were stained by haematoxylin and eosin. Hepatic glycogen were observed after PAS stainning. Genes in insulin signaling pathway as IRS-1、IRS-2、Akt、PKC、and glucose and lipid metabolism related genes AMPKα, PPARα, GSK-3α, SREBP-1c, SREBP-2, FAS mRNA expression in hepatic tissue were detected by real-time PCR.Inflammation-associated cytokines NF-κB、TNF-α、IL-1β and endoplasmic reticulum stress (ERS) pathway related indicators such as eIF2α、ATF4、CHOP、IRE1α、XBP1 and GRP78 rriRNA expression in hepatic tissue were detected by real-time PCR; Immunohistochemically method was performed with p-eIF2α、GRP78 and CHOP antibody; Western Blotting was performed to detected p-eIF2aand GRP78 proteins expression in hepatic tissue.ResultsEach doses JTXKG had no significant effect on food intake in KK-Ay mice. The body weight of all mice were continuely rising; high and medium doses JTXKG reduced liver/body weight of KK-Ay significantly (P<0.01).Medium dose JTXKG relieved lymphocytes infiltration, sinusoidal space dilation and micro vesicular steatosis in liver tissue.JTXKG lowered fasting blood glucose in 10 weeks treatment (P<0.01). Glucose tolerance had no change at week 4 (P>0.05), but showed a significant improvement in high dose JTXKG group at week 10 (P<0.01). High and medium doses JTXKG could decrease serum FINS, HbAlc and improve ISI (P<0.01). Low dose JTXKG could decrease serum FINS and improve ISI (P< 0.05). Medium dose JTXKG could improve glycogen reservation, high and medium doses JTXKG could decrease the mRNA expression of hepatic GSK-3a effectively (P<0.01).In the aspect of serum lipid contents, high dose JTXKG could decrease serum LDL and TG (P<0.01), medium dose JTXKG decreased serum TC, TG, HDL, LDL (P<0.01) and FFA (P<0.05). Low dose JTXKG decreased serum TC, FFA, HDL, LDL (P<0.01). In the aspect of hepatic lipid contents, Low dose JTXKG could decrease TG and improve HDL (P <0.01). High and medium doses JTXKG could decrease hepatic TC, TG, LDL and improve HDL (P<0.05) and the effect of medium dose is better than the high dose. In the mRNA expression of glucose and lipid metabolism, High dose JTXKG could improve the mRNA expression of PPARa and reduce the genes expression of SREBP1c (P<0.01). High dose JTXKG could improve the mRNA expression of PPARa, Insig-1 (P<0.05) and decrease the genes expression of SREBP1c. SREBP2 (P<0.05) and FAS (P<0.01). Low dose JTXKG could improve the mRNA expression of AMPKa (P<0.01) and decrease the genes expression of SREBPlc, SREBP2 significantly (P<0.01).All doses JTXKG could improve the mRNA expression of IRS-2, medium dose JTXKG also can improve the mRNA expression of Akt (P<0.01).Liver function:Each dose of JTXKG could significantly reduce serum ALT and y-GT (P<0.01). High dose JTXKG reduce serum AST significantly (P<0.01). Medium and low doses JTXKG have no significant effect on serum AST (P>0.05).Oxidative stress:In the aspect of serum SOD and MDA contents, all JTXKG decrease MDA and increase SOD activity (P<0.01) and medium dose JTXKG is most effective. In the aspect of hepatic MDA^ SOD and GSH contents, high and medium doses JTXKG decrease MDA content and increase SOD activity effectively (P<0.01), GSH activity is enhanced at the same time (P<0.05), effect of high dose JTXKG is better than the medium one, but the difference is not significant (P>0.05). Low dose JTXKG is not effective in mitigating oxidative stress (P>0.05).Inflammation-associated cytokines:Medium and low doses JTXKG reduce the genes expression of NF-κB (P<0.01) and TNF-a(P<0.05). All doses of JTXKG reduce the mRNA expression of IL-1β, but the difference is not significant (P>0.05).ERS pathway:In the mRNA expression of indicators related to ERS pathway, high dose JTXKG reduce the mRNA expression of eIF2a and ATF4 (P<0.01), but ineffective with genes expression of CHOP、IRE1α XBP1 and GRP78 (P>0.05). Medium dose JTXKG reduce the mRNA expression of eIF2α、ATF4、GRP78 (P<0.01) and CHOP、 IRE1α (P<0.05), but ineffectiveness in gene expression of XBP1 (P>0.05). Low dose JTXKG reduce the mRNA expression of ATF4 (P<0.01) and GRP78 (P<0.05), but ineffectiveness in genes expression of CHOP、IRE1α XBP1 and GRP78 (P>0.05). All doses JTXKG can reduce the phosphorylation level of eIF2a and the proteins expression of GRP78 and CHOP (P<0.01)Conclusions1 JTXKG can reduce the blood glucose levels, regulate glucose metabolism, improve the insulin sensitivity, and maintain glucose homeostasis of the T2DM mice. High and medium doses JTXKG are most effective.2 JTXKG can decrease serum and hepatic lipid profiles and protect liver from being damaged by T2DM condition of the T2DM mice. High and medium doses JTXKG are most effective.3 JTXKG can regulate hepatic glucose metabolism of the T2DM mice by upregulating the gene expression of IRS-2.4 JTXKG can upregulate the gene expression of AMPKa accordingly to regulate the genes expression of PPARa, SREBPs, FAS and Insig-1 that related to lipid metabolism, through which to regulate lipid metabolism and relieve hepatic steatosis.5 JTXKG can mitigate systemic oxidative stress and improve the body’s antioxidant ability, to ensure the normal transmission of insulin signal systematically. High and medium doses JTXKG are most effective.6 JTXKG has an overall anti-inflammatory effects and can inhibit the activation of NF-kappa B inflammatory signaling pathways, reduce the secretion of inflammatory cytokines to increase the phosphorylation of IRS tyrosine, improve insulin sensitivity and reduce systemic oxidative stress. Medium and low doses JTXKG are most effective.7 JTXKG can alleviate the endoplasmic reticulum stress, ensure the normal intracellular insulin signaling transmission, improve insulin physiological function and reduce insulin resistance in T2DM. Medium dose JTXKG is most effective.This research adopts the high fat feed induced spontaneous T2DM KK-Ay mice model to probe the protective effect of JTXKG to diabetic liver injury and the improvement on hypoglycemic and glucolipid metabolic disorders. From the relationship between IR, liver oxidative stress, inflammation, and endoplasmic reticulum to explore the possible mechanism of JTXKG improve diabetic liver injury.