| Objective:1.To investigate the effects of 2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione,isolated?DMDD?from the roots of Averrhoa carambola L.?Oxalidaceae?on palmitic acid induced human skeletal muscle cell?HSKMC3500?insulin resistance.To clarify whether DMDD could be improved the insulin resistance of skeletal muscle cells by inhibiting TLR4 signal transduction pathway.2.To investigate the effects of DMDD isolated from the roots of Averrhoa carambola on high-fat,high-sugar diet plus STZ induced insulin resistance in TLR4 gene knockout diabetic mice.Methods:1.In this study,HSKMC cell is divided into six groups:Normal group?Normal group?,model group?PA group?,positive control group ?docosahexaenoic acid?DHA?group?,DMDD High,Mid and Low dose group?DMDD High,DMDD Mid,and DMD Low group?.1.1.MTT assay was used to determine the concentration of PA,in HSKMC cell,the concentration of DHA in HSKMC cells and the concentration of DMDD in HSKMC cells.1.2.Effect of DMDD on inflammatory factor expression in HSKMC cell:Quantitative real-time PCR was used to detect the mRNA leves of Toll-like Receptor 4?TLR4?and Myeloid Differentiation Factor 88?MyD88?,Nuclear Factor KB?NF-?B?,Tumor necrosis Factor alpha?TNF-a?and Interleukin–6?IL-6?,Monocyte Chemotactic Protein 1?MCP-1?mRNA expression. Western Blot method was used to detect the protein expressions of TLR4,MyD88,nuclear phosphorylation NF-?B p65?NF-?B p65?,TNF-a and McP-1protein.2.In this study,six to eight weeks old male TLR4 gene knockout C57BL/10 mice and TLR4 wild type FVB mice were randomly divided into two groups:normal diet?Normal?group and high-sugar,high-fat diet?HFHSD?group,HFHSD group were fed a high-fat,high-sugar diet for 4 weeks,after that the mice were injected with STZ?120 mg/kg body weight?via a tail vein.After 72 h,the mice with FBG?11.1 mmol/L were confirmed as having diabetes.Then were divided into eight groups:FVB normal group,FVB control group,C57BL/10 normal group,C57BL/10 model group,pioglitazone?PIO?group,DMDD low dose?DMDD low?group,DMDD middle dose?DMDD Mid?group,and DMDD high dose?DMDD High?group.These animals were orally administered DMDD(12.5,25.0,or 50 mg·kg-1 of body weight per day),pioglitazone?30 mg/kg of body weight per day?and the mice in FVB control group were orally administered DMDD(50 mg·kg-1 of body weight per day)once a day for 4 weeks,and the mice in C57BL/10 normal group,FVB normal group and C57BL/10 model group received an equal volume of distilled deionized water.Body weight were monitored weekly and FBG were monitored every two weeks.The 28 days proceed Oral glucose tolerance test?OGTT?,the35 days proceed Insulin tolerance test?ITT?and the 42 days proceed Insulin resistance test?IRT?.After the 49 days of treatment,the mice were fasted overnight,and blood was then collected from the retro-orbital venous sinus.Subsequently,the mice were executed,and the following indicators were measured:2.1.The level of serum total cholesterol?TC?,triglyceride?TG?,low density lipoprotein?LDL-C?,high density lipoprotein?HDL-C?,Alanine aminotransferase,Alanine transaminase,ALT),Aspartate aminotransferase,?Aspartate,AST?and Alkaline phosphatase,Alkaline phosphatase,ALP)were measured by biochemical analyzer;2.2.The levels of serum Insulin?Insulin?,free fatty acid?FFA?,tumor necrosis factor alpha?TNF alpha?,interleukin 6?IL-6?,Leptin,Leptin,Resistin,Resistin and adiponectin?ADP?,Insulin resistance index?IRI?,impaired glucose tolerance?OGTT?and Insulin tolerance?ITT?,Insulin release test?IRT?were measured by enzyme-linked immunosorbent?ELISA?.;2.3.The content of malondialdehyde?MDA?and the activities of superoxide dismutase?SOD?in liver homogenates were determined by ELISA.2.4.The pathological changes of liver and pancreas tissue were observed by regular Hematoxylin-eosin?HE?staining;The protein expressions of Toll-like receptor 4?TLR4?,Myeloid differentiation factor 88?MyD88?,Nuclear factor-kappa B?NF-?B?,Cysteinyl aspartate specific proteinase-3?Caspase-3?,Cysteinyl aspartate specific proteinase-8?Caspase-8?,Cysteinyl aspartate specific proteinase-9?Caspase-9?,Bcl-associated X protein?Bax?and B-celllymphoma-2?Bcl-2?in the liver and pancreas tissue were analyzed by immunohistochemistry;Meanwhile,terminal-deoxynucleoitidyl transferase mediated nick end labeling?TUNEL?staining was used to determine the number of positive cells in pancreas and hepatocytes samples;2.5.The mRNA expressions of TLR4,MyD88,NF-?B,TNF-?and IL-6 in liver and pancreas tissue were evaluated using Real-time quantitative polymerase chain reaction?RTQ-PCR?.2.6.The pancreas and liver in mice were observed by electron microscopy.2.7.TUNEL method was used to detect the apoptosis of islet cells and hepatocytes.Results:1.Compared with PA group,the glucose concentration significantly was reduced in DMDD High group,DMDD Mid,DMDD Low group and DHA group?P<0.01?.DMDD can reduce PA induced HSKMC cells insulin resistance.2.Compared with the PA group,the mRNA expressions of TLR4,MyD88,NF-?B,IL-6,TNF-a,MCP-1 mRNA levels were reduced in DMDD High group,DMDD Mid,DMDD Low group and DHA group were down-regulated?p<0.05 or p<0.01?,and there's a dose relationship.3.Compared with the PA group,the protein expressions of TLR4,MyD88,NF-?B,TNF-a,MCP-1 protein down-regulated in DMDD High group,DMDD Mid,DMDD Low group and DHA group were down-regulated?p<0.05 or p<0.01 or p<0.001?.4.Compared with the FVB normal group and C57BL/10 normal group, FVB control group were detected that DMDD didn't have obvious toxic effects.5.Compared with the C57BL/10 model group,the body weight,FBG,serum levels of TC,TG,TC,TG,LDL-C,ALT,AST and ALP were decreased by DMDD?P<0.01?,while HDL-C increased?P<0.01?;6.Compared with the C57BL/10 model group,the serum levels of Insulin,FFA,TNF-?,IL-6,LEP and Resistin were decreased by DMDD;7.The content of MDA in liver homogenates were decreased by DMDD?P<0.01?,while the activities of SOD in liver homogenates were increased by DMDD.8.RTQ-PCR analysis showed that the mRNA expression levels of MyD88,NF-?B,TNF-?and IL-6 were up-regulated in the liver and pancreatic tissues of the C57BL/10 model group compared with the levels observed in the FVB normal group,FVB control group and C57BL/10 normal group.After the DMDD and PIO treatment,the mRNA expression levels of MyD88,NF-?B,TNF-?and IL-6 were down-regulated.9.HE staining demonstrated that significant improvement in hepatic fat degeneration in the treatment group.Compared with the C57BL/10 model group,after treatments with PIO and DMDD,the cytoarchitecture of pancreatic islet cells were effectively recovered.10.The results of electron microscopy showed that compared with the C57BL/10 model group,the mitochondrial damage in the liver tissue was decreased,the endoparasitic reticulum was improved,and the mitochondria were increased;Mitochondrial damage decreased in pancreatic tissue,improved endoparasitic reticulum,and increased beta cells and secretory granules by DMDD treatment group and the PIO group.11.Immunohistochemical analysis showed that the liver and pancreatic tissue expression levels of MyD88,NF-?B and the expressions of apoptosis-related regulators?including caspase-3,caspase-8 and caspase-9?and the apoptosis-induced protein Bax were markedly down-regulated by DMDD,while the expression of the anti-apoptotic Bcl-2 protein was notably increased by DMDD.Compared with FVB normal group,FVB control group and C57BL/10normal group,the number of apoptotic islet cells was increased significantly in C57BL/10 model group.Compared with C57BL/10 model group,the number of islet cell apoptosis were decreased significantly by DMDD treatment group.In addition,the number of TUNEL-positive cells in C57BL/10 model group markedly increased compared with that in the FVB normal group,FVB control group and C57BL/10 normal group,while the mice in DMDD treatment group showed a significant decrease in cells positively stained by TUNEL when compared with the C57BL/10 model group.Conclusions:1.DMDD may be improve PA induced HSKMC cells insulin resistance through TLR4/NF-?B signaling pathway.2.The study shows that DMDD was not toxic to mice when use it for a long time.DMDD did not through TLR4 signaling transduction pathways then were improved insulin resistance in diabetes mice by NF-?B signaling transduction pathways.It also prompts DMDD were improved the diversity of the insulin resistance mechanism.However we sure DMDD through reduced FFA,then improved insulin resistance,thus effect may be linked to the improvement of hyperglycemia,hyperlipidemia,antioxidation,anti-inflammatory,and inhibition of apoptosis.The molecular mechanism needs to research in the future. |
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