| Backgrounds: Type 2 diabetes mellitus(T2DM)is an independent and increased risk factor for non-alcoholic fatty liver disease(NAFLD).It has been demonstrated that ox LDL levels are significantly elevated in patients with T2 DM and promotes the occurrence and development of NAFLD by accelerating the oxidative stress and microcirculatory dysfunction in liver endothelial cells.Liver sinusoids endothelial cell(LSEC)is one of the main cells that maintain hepatic sinusoidal structure and function and regulate hepatic sinusoidal contraction.The structural and functional change of LSECs was the foundation of sinusoidal capillarization,which is the main pathological feature of type 2 diabetes combined with NAFLD.Integrin ?v?5 is one of the cell adhesion molecules,which play an important role in the cell inflammatory,adhesion,activation,proliferation,and so on.The interaction between intergrin av?5 and its binding vitronectin(Vn)plays an important role in diabetic fatty liver disease.FAK/ Rho GTPase combined with integrins to cause a series of cascade amplification effects,and ultimately regulate cell function.However,it remained unclear whether integrin av?5 influenced the sinusoidal capillarization by adjusting Vn and FAK/Rho GTPase and was involved in the pathogenesis of T2 DM with NAFLD,and whether GLP-1 can influence integrin av?5 expression and integrin av?5 induced sinusoidal capillarization in diabetic fatty liver disease.And it has not been reported at home and abroad.Objective and significance: we aimed to investigate the role and signaling pathways of integrin av?5 in the sinusoidal capillarization induced by high glucose and oxLDL at the cellular level,and to further validate our hypothesis by establishing a rat model of T2 DM with NAFLD from the overall level.This study will help to elucidate the role of integrin av?5 in diabetics with fatty liver and the pathogenesis of diabetic fatty liver.It provides a theoretical basis for the future use of integrin av?5 as a new target for the prevention and treatment of diabetes with fatty liver and to explore new intervention pathways.Methods: Firstly,we analyzed the effects of the different concentrations of high glucose or ox LDL on the expression of integrin av?5 and Vn in the different time by real-time RT-PCR,and then we established diabetic fatty liver rat model by high-fat feed and intraperitoneal injection of low dose of STZ.Biochemical method,light microscopy,and transmitting electronic microscopy were used to observe metabolic changes,pathological features,and ultrastructure of liver sinusoids in diabetic fatty liver rats,respectively.Immunohistochemistry was used to detect the differential expression and distribution of integrin av?5 in liver tissues of normal control group and diabetic fatty liver group.Secondly,the expression of the integrin av?5 gene in liver sinusoidal endothelial cells was silenced using cell transfection and RNA interference(RNAi).The changes of fenestrae of the HLSECs before and after silencing of integrin av?5 gene were observed by scanning electron microscope.The expression and distribution of basement membrane protein Vn before and after integrin av?5 silencing were analyzed by RT-PCR,western blotting,immunofluorescence and laser confocal microscopy.Thus,we further revealed the role of integrin av?5 in high glucose and oxLDL-induced hepatic sinusoidal capillarization.RT-PCR,Western blotting and immunofluorescence were used to detect the mRNA and protein expressions of candidate downstream pathway genes after integrin av?5 gene silencing,to compare to the changes of key signaling pathway molecules in the experimental and control groups,and to reveal the mechanism of integrin av?5 gene in hepatic sinusoidal capillarization from the molecular level.Next,HE staining,Masson staining,Sirius red staining,immunohistochemistry,and projection electron microscopy were performed to analyze the effect of integrin av?5 inhibitor on the morphology and function of hepatic tissue and sinusoidal capillarization in diabetic fatty liver rats.Finally,the effects of GLP-1 on the expression of integrin av?5 were analyzed by ELISA and Western blotting.The effects of GLP-1 on integrin av?5 induced sinusoidal capillarization were detected by immunohistochemistry and electron microscopy.Results:(1)The results suggested that not only high glucose but also ox LDL can upregulate the expression of integrin av?5 and Vn in time-and concentration–dependent manners,and then stimulate hepatic sinusoid capillarization in HLSECs.More importantly,both RT-PCR and Western blotting results revealed that the mRNA and protein expression of integrin av?5 and Vn markedly increased in the high glucose and ox LDL group compared with the NC or ox LDL group at 24 h(P < 0.05);(2)In the model group of diabetic fatty liver rats,the liver volume was significantly increased,the capsule was tight,and the entire liver was yellow-white.HE staining showed that there were a large number of lipid droplets in the cytoplasm of hepatocytes,and most liver cells showed fatty degeneration.Masson's staining and Sirius red staining showed a large number of collagen fibrosis tissues with obvious fiber proliferation.The study observed the ultrastructure of liver tissue using TEM,and found that: the mitochondria(M)structure was partly swollen,vacuolar were degenerated,smooth endoplasmic reticulum(ER)proliferated,and rough endoplasmic reticulum expanded,the space of Disse widened,the basement membrane(BM)thickened and became continuous(P <0.05);(3)Integrin av?5 was mainly localized and distributed in the nucleus and cytoplasm of liver sinusoidal endothelial cells in the diabetic fatty liver by IHC,Moreover,the expression level of integrin av?5 in diabetes with fatty liver was significantly higher than that in the healthy control group(P < 0.05).Therefore,we speculate that integrin av?5 may participate in the pathogenesis of diabetic fatty liver;(4)Using scanning electron microscopy,the study observed the diameter and number of fenestrae of HLSECs,and found that HLSECs were porous,and the basement membrane was discontinuous in the normal control group.However,the diameter and number of fenestrae of HLSECs in the high glucose and oxLDL group are significantly less than the control group(P <0.05).More interestingly,after silence of integrin ?v?5,the number of the fenestrae increased significantly,and the diameter significantly became larger(P <0.05);(5)Intergrin av?5 can enhance high glucose and oxLDL-induced sinusoidal capillarization(the decrease of the diameter and number of the fenestrae and the increase of Vn),while knockdown of intergrin av?5 markedly attenuated these effects;(6)The expressions of FAK,RhoA and Rac1 increased in high glucose and oxLDL,but the expressions of FAK,RhoA and Rac1 decreased after silence of integrin ?v?5.We speculated that integrin ?v?5 promotes hepatic sinusoidal capillarization,and its mechanism may be achieved through the activation of the FAK/Rho GTPase signaling pathway;(7)Compared to the NC or NAFLD groups,FAK,RhoA,and Rac1 protein expressions were significantly increased in the T2DM-NAFLD group(P < 0.05),and after the treatment of the integrin av?5 inhibitor,FAK,RhoA,and RAC1 were remarkably decreased(P < 0.05).More interestingly,the mRNA results of FAK,RhoA,and Rac1 by RT-PCR were consistent with the protein results by IHC;(8)The levels of ALT,AST,TC,TG,fasting insulin,HOMA-IR and liver coefficient were significantly increased in the T2DM-NAFLD group compared with the NC or NAFLD group(P<0.05),However,after the integrin av?5 inhibitor treatment,the levels of the above-mentioned indicators were markedly decreased(P < 0.05);(9)In the rat model study,there was a significant increase in Vn,collagen fibers(CF),space of Disse and basement membrane thickness in the rats' livers in the T2DM-NAFLD group compared with the NC or the NAFLD group.More importantly,the intergrin av?5 inhibitor can markedly reverse the above-mentioned hepatic damage in the diabetic rat model with NAFLD.Integrin av?5 inhibitor not only alleviated the progression of hepatic sinus dysfunction,but also improved liver morphology and function damage in vivo,and it played a significant antifibrotic effect in a diabetic fatty liver rat model;(10)GLP-1 not only can inhibit the expression of integrin ?v?5 in liver tissues,cells and serum,but also can inhibit integrin ?v?5-induced hepatic sinusoidal capillarization.Conclusions: Integrin av?5 activates the FAK/Rho GTPase signaling pathway by interacting with Vn,enhancing sinusoidal capillarization induced by high glucose and ox LDL,and aggravating hepatic injury in diabetic rats with NAFLD,thereby participating in the pathogenesis of diabetic fatty liver disease.GLP-1 plays a role in the protection of diabetes with fatty liver by inhibiting the expression of integrin av?5 and integrin av?5 induced sinusoidal capillarization.Integrin av?5 may be one of the new targets for the treatment of diabetic fatty liver by GLP-1. |
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