第一临床医学院知识库

研究背景：2型糖尿病（T2DM）是非酒精性脂肪肝（NAFLD）独立的危险因素。2型糖尿病患者体内oxLDL水平明显升高，加速氧化过程，是导致肝脏微循环功能障碍的重要触发因素，促进了NAFLD的发生发展。肝窦内皮细胞(liver sinusoids endothelial cell, LSEC)是维护肝窦结构功能和调节肝窦收缩的主要细胞之一，其结构和功能的改变导致肝窦毛细血管化，而肝窦毛细血管化是2型糖尿病合并脂肪肝的主要病理特征。整合素αvβ5是一种细胞粘附分子，在细胞炎症、活化和增殖中起着重要的作用，它与其配体玻连蛋白（Vn）之间的相互作用在糖尿病性脂肪肝中起着重要作用。FAK/Rho GTPase与整合素结合后引起一系列级联放大效应，最终调节细胞功能。然而，整合素avβ5是否通过调控FAK/Rho GTPase信号通路影响肝窦毛细血管化，从而参与糖尿病合并脂肪肝的发病机制？胰高血糖素样肽1(GLP-1)受体激动剂艾塞那肽是否通过影响整合素avβ5的表达和整合素avβ5诱导的肝窦毛细血管化，从而达到缓解脂肪肝的目的？目前仍然不清楚，国内外未见报道。

目的及意义：本研究旨在细胞水平上探讨整合素avβ5在高糖和oxLDL诱导的肝窦毛细血管化中的作用及其信号途径；并通过建立糖尿病合并脂肪肝大鼠模型，从整体水平上进一步验证我们的假说。本研究将有助于阐明整合素avβ5在糖尿病合并脂肪肝中的作用和糖尿病性脂肪肝的病理发生机制，为今后将整合素avβ5作为防治糖尿病合并脂肪肝的新的靶标和探索新的干预途径提供理论依据。

方法：首先采用实时定量PCR反应（RT-PCR）、Western blotting方法分别检测不同浓度高糖或oxLDL对整合素avβ5、Vn mRNA及蛋白表达的影响，利用高脂饲料喂养结合小剂量链脲佐菌素（STZ）腹腔注射建立糖尿病性脂肪肝大鼠模型，采用生化法、光镜和投射电镜分别观测糖尿病性脂肪肝大鼠的代谢指标变化、病理特点和肝窦的超微结构，通过免疫组化检测整合素avβ5在正常对照组和糖尿病性脂肪肝组肝组织中的差异表达和分布情况；其次，应用细胞转染、RNA干扰技术（RNAi）靶向沉默肝窦内皮细胞整合素avβ5基因的表达，通过扫描电镜观测整合素avβ5基因沉默前后肝窦内皮细胞窗孔的变化，通过RT-PCR、Western blotting、免疫荧光、激光共聚焦等技术分析整合素avβ5沉默前后基底膜蛋白Vn的表达变化及分布情况，进一步揭示整合素avβ5在高糖和oxLDL诱导的肝窦毛细血管化中的作用，应用RT-PCR、Western blotting和免疫荧光检测整合素avβ5基因沉默后部分候选下游通路基因mRNA及蛋白的表达，比较实验组和对照组中关键信号通路分子的变化，从分子水平揭示整合素avβ5基因在肝窦毛细血管化中的作用机制；接下来，采用HE染色、Masson染色、天狼星红染色、免疫组化、投射电镜等分析整合素avβ5抑制剂对糖尿病性脂肪肝大鼠肝组织形态和功能以及肝窦毛细血管化的影响；最后通过ELISA法、Western blotting分析GLP-1对整合素avβ5表达的影响，使用免疫组化、电镜等方法检测GLP-1对整合素avβ5诱导的肝窦毛细血管化的影响。

结果：(1) 高糖以时间和浓度依赖的方式上调整合素avβ5和Vn的表达；同样地，oxLDL也以时间和浓度依赖的方式促进整合素avβ5和Vn的表达，从而诱导HLSECs的肝窦毛细血管化。更重要的是，RT-PCR和Western blotting结果显示，与NC组或oxLDL组相比，高糖和oxLDL组整合素avβ5和Vn的mRNA和蛋白表达水平显著增加(P < 0.05)；(2) 与正常对照组相比，糖尿病合并脂肪肝模型组大鼠肉眼大体可见肝脏体积明显增大，包膜紧张，整个肝脏呈黄白色，光镜下HE染色可见肝细胞胞浆内出现大量的脂滴，大部分肝细胞呈脂肪变性，Masson染色及天狼星红染色可见大量胶原纤维组织沉积，纤维增生明显，投射电镜可见肝细胞器中线粒体肿胀，粗面内质网增生，滑面内质网扩张，Disse间隙增宽；肝窦内皮细胞肿胀增生，基底膜增厚，呈连续的基底膜；(3) 整合素avβ5主要定位和分布在糖尿病合并脂肪肝组肝脏的肝窦内皮细胞的胞核和胞浆中，而且，整合素avβ5在糖尿病合并脂肪肝组的表达水平明显高于健康对照组。因此，我们推测整合素avβ5可能参与了糖尿病性脂肪肝的发病机制；(4) 采用扫描电镜观察了HLSECs窗孔的直径和数量，结果发现：在正常对照组，HLSECs是多孔的，基底膜是不连续的，然而，在高糖和oxLDL组，HLSECs窗孔的直径和数量明显小于或少于对照组(P<0.05)；更有趣的是，沉默整合素avβ5后窗孔数量明显增多了，直径反而变大了(P< 0.05)；(5) 整合素avβ5能诱导肝窦毛细血管化（窗孔直径和数量的减少以及Vn的增加），沉默或抑制整合素avβ5会显著降低这一效应；(6) 高糖和oxLDL刺激下HLSECs表达FAK、RhoA和Rac1增多，但整合素αvβ5沉默后FAK、RhoA和Rac1表达反而减少。我们推测整合素αvβ5促进肝窦毛细血管化，其机制可能通过激活FAK/Rho GTPase信号途径而完成的；(7) 与NC或NAFLD组相比，T2DM+NAFLD组大鼠肝组织FAK，RhoA和Rac1蛋白表达显著增加(P<0.05)，而使用整合素αvβ5抑制剂治疗后，FAK、RhoA和RAC1表达明显降低(P <0.05)。更有趣的是，采用RT-PCR得到的FAK、RhoA和Rac1的mRNA结果与IHC的检测结果一致(P <0.05)；(8) 与NAFLD或NC组相比，T2DM+NAFLD模型组大鼠血清ALT、AST、TC、TG、空腹胰岛素水平以及胰岛素抵抗指数、肝脏系数显著升高(P < 0.05)，而使用整合素αvβ5抑制剂治疗4周后，上述指标明显下降(P < 0.05)；(9) 在大鼠模型中，与正常对照组相比，糖尿病合并脂肪肝组大鼠肝脏基底膜蛋白（Vn）、胶原纤维、Disse间隙和基底膜厚度明显增加(P < 0.05)。更重要的是，整合素αvβ5抑制剂可显著逆转上述肝损伤。在体内整合素avβ5抑制剂不仅缓解了肝窦功能障碍的进展，而且改善了肝脏形态和功能的损伤，并在糖尿病性脂肪肝大鼠模型中发挥了明显的抗纤维化作用；(10) GLP-1不仅能够抑制肝组织、细胞和血清中整合素αvβ5的表达，而且能够抑制整合素αvβ5诱导的肝窦毛细血管化。

结论：整合素avβ5通过与Vn相互作用激活FAK/Rho GTPase信号通路，增强高糖和oxLDL诱导的肝窦毛细血管化，并加重糖尿病合并脂肪肝大鼠的肝损伤，从而参与了糖尿病性脂肪肝的发病机制。GLP-1通过抑制整合素avβ5表达和整合素avβ5诱导的肝窦毛细血管化而发挥保护糖尿病合并脂肪肝的作用。整合素avβ5有可能成为GLP-1治疗糖尿病性脂肪肝的新的作用靶点之一。

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 oxLDL levels are significantly elevated in patients with T2DM 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 αvβ5 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 avβ5 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 avβ5 influenced the sinusoidal capillarization by adjusting Vn and FAK/Rho GTPase and was involved in the pathogenesis of T2DM with NAFLD, and whether GLP-1 can influence integrin avβ5 expression and integrin avβ5 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 avβ5 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 T2DM with NAFLD from the overall level. This study will help to elucidate the role of integrin avβ5 in diabetics with fatty liver and the pathogenesis of diabetic fatty liver. It provides a theoretical basis for the future use of integrin avβ5 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 oxLDL on the expression of integrin avβ5 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 avβ5 in liver tissues of normal control group and diabetic fatty liver group. Secondly, the expression of the integrin avβ5 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 avβ5 gene were observed by scanning electron microscope. The expression and distribution of basement membrane protein Vn before and after integrin avβ5 silencing were analyzed by RT-PCR, western blotting, immunofluorescence and laser confocal microscopy. Thus, we further revealed the role of integrin avβ5 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 avβ5 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 avβ5 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 avβ5 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 avβ5 were analyzed by ELISA and Western blotting. The effects of GLP-1 on integrin avβ5 induced sinusoidal capillarization were detected by immunohistochemistry and electron microscopy.

Results: (1) The results suggested that not only high glucose but also oxLDL can upregulate the expression of integrin avβ5 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 avβ5 and Vn markedly increased in the high glucose and oxLDL group compared with the NC or oxLDL 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 avβ5 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 avβ5 in diabetes with fatty liver was significantly higher than that in the healthy control group (P < 0.05). Therefore, we speculate that integrin avβ5 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 αvβ5, the number of the fenestrae increased significantly, and the diameter significantly became larger (P <0.05); (5) Intergrin avβ5 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 avβ5 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 αvβ5. We speculated that integrin αvβ5 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 avβ5 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 avβ5 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 avβ5 inhibitor can markedly reverse the above-mentioned hepatic damage in the diabetic rat model with NAFLD. Integrin avβ5 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 αvβ5 in liver tissues, cells and serum, but also can inhibit integrin αvβ5-induced hepatic sinusoidal capillarization.

Conclusions: Integrin avβ5 activates the FAK/Rho GTPase signaling pathway by interacting with Vn, enhancing sinusoidal capillarization induced by high glucose and oxLDL, 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 avβ5 and integrin avβ5 induced sinusoidal capillarization. Integrin avβ5 may be one of the new targets for the treatment of diabetic fatty liver by GLP-1.