| Background: Diabetes is one of the most important non-communicable diseases that threaten the health of all humans.It is estimated that the incidence of diabetes in China is as high as 11.6%.Cardiovascular complication is the leading cause of death in diabetic patients and has become the focus of clinical prevention and treatment.Chronic low-grade inflammation and activation of the innate immune system play a major role in the field of diabetes research.The TOLL-like receptor pathway is the first line of defense for innate immunity,and it is also a bridge connecting innate immune and inflammation.Studies have been reported that inappropriate activation of TLR4 and inflammation pathways are involved in the occurrence and development of diabetic cardiovascular complications.Liraglutide,a novel hypoglycemic agent,has been reported to inhibit the release of inflammatory factors.A number of large clinical studies have confirmed that liraglutide has cardiovascular protection properties,but exact mechanisms are not yet clear.Objective: To investigate whether liraglutide can protect the cardiovascular system of diabetic rats by down-regulating TLR4-mediated inflammatory pathways in vivo and in vitro.Methods: Eight-week-old male Sprague-Dawley rats were randomly divided into two groups,normal control group and diabetic model group;the rats in normal control group were fed with chow diet and those of diabetic model group were fed with high-fat diet.After 4 weeks,the rats in diabetic model group were treated with tail vein injection of streptozotocin(45 mg/kg)to make diabetic rat models.One week later,the diabetic rats were randomly divided into two groups: simple diabetic group(group D),intraperitoneal injection of normal saline 1ml/kg/d;liraglutide treatment group(group G),intraperitoneal injection of Liraglutide 1ml/kg/d;while the normal control group rats(group N),intraperitoneal injection of normal saline 1ml/kg/d.After 16 weeks,all the rats were executed through femoral artery bloodletting,and blood samples were taken to measure blood glucose,blood lipids,liver and kidney function,and C-reactive protein.We observed the pathomorphology of aorta and myocardium,the changes of TLR4,My D88 and NF-?B p65 in aorta and myocardium by immunohistochemistry and detected the changes of m RNA and protein expression levels of TLR4,My D88,NF-?B p65 in each group by q RT-PCR and Western Blot methods in vitro experiments.A7R5 rat vascular smooth muscle cells were used.According to the culture medium and intervention methods,they were divided into: low-glucose culture group(5mmol/L,L group),high-glucose culture group(25mmol/L,H group),low-glucose culture + liraglutide treatment group(100 n M,LG group),high-glucose culture + liraglutide treatment group(100 n M,HG group),low-glucose culture + liraglutide + exendin(ex9-39,100 n M)treatment group(LGE group),high-glucose culture + liraglutide + exendin(100 n M)treatment group(HGE group),cultured for 12 h and 24 h,respectively.The expression of TLR4,My D88 and NF-?B p65 in vascular smooth muscle cells of each group was observed by immunofluorescence staining at 24 h.RT-PCR and Western blot methods were used to detect the changes of m RNA and protein expression levels of TLR4,My D88 and NF-?B p65 in vascular smooth muscle cells at 12 h and 24 h in each group.Changes of Myd88 and NF-?B p65 m RNA and protein expression levels were detected.Data were analysed by one-way ANOVA.Results: 1.Presentation of blood biochemical parameters of rats in each group: Through high-fat feeding combined with tail vein injection of STZ,blood glucose of rats was significantly higher than that of the normal group,suggesting diabetic rats model was successfully established.The transaminase,triglyceride and cholesterol in group D were significantly elevated than those in group N and G.There was no significant difference in transaminase,triglyceride and cholesterol between group G and group N.Compared with group N,the levels of C-reactive protein in group D and group G were significantly increased.Compared with group D,the level of C-reactive protein in group G was significantly decreased.2.The pathological changes of rats in each group: The pathological appearance of the aorta in group D was rough intimal,the endothelial cells hyperplasia,smooth muscle cells hypertrophy and disordered arrangement.Degeneration and necrosis of cardiomyocytes were observed.Inflammatory cell infiltration and fibrosis could occur.Immunohistochemistry results: In group D,rat aortic endothelial cells and smooth muscle cells and myocardial cell membrane and cytoplasm of TLR4,My D88,cytoplasm and nucleus of NF-?B p65 were highly expressed,with strong brown-yellow staining;Group G rats showed reduced pigmentation,which was between N and D groups.3.Results of PCR and Western Blot in aorta and myocardium: The expression of TLR4,My D88 and NF-?B p65 in aorta and myocardial tissue of group D were increased;the expression of group G was significantly lower than that of group D.4.In vitro cell experiments: Compared with group L,the expression of TLR4,My D88 and NF-?B p65 in group H was significantly increased.After intervention with liraglutide,the expressions of TLR4,My D88 and NF-?B p65 in HG and LG groups were significantly lower than those in H group and L group respectively;after intervention with liraglutide and exendin(9-39),the expressions of TLR4,My D88,and NF-?B p65 in HGE and LGE groups were significantly higher than those in HG and LG groups,respectively.Conclusion: The expression of TLR4,My D88 and NF-?B p65 were highly expressed in the aorta and heart of diabetic rats,indicating that the activation of TLR4 pathway and its mediated inflammatory process participate in the pathogenesis of diabetic cardiovascular complications.Liraglutide can reduce the pathological changes of the cardiovascular system in diabetic rats by down-regulating the TLR4-mediated inflammation pathway in the aorta and myocardium and reducing the expression of inflammatory factors,thus protecting the cardiovascular system of diabetic rats... |
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