糖脂清方改善胞葬过程中细胞骨架重排缓解糖尿病动脉粥样硬化的机制研究

Mechanism research of Tangzhiqing Formula in alleviating diabetic atherosclerosis by improving cytoskeleton rearrangement in efferocytosis

关键词:  糖尿病动脉粥样硬化  胞葬  细胞骨架重排  糖脂清方

Key words:  diabetic atherosclerosis  efferocytosis  cytoskeleton rearrangement  Tangzhiqing Formula

摘要:

[目的] 探究糖脂清方（TZQ）治疗糖尿病动脉粥样硬化的作用机制。[方法] 36只雄性ApoE^-/-小鼠使用高脂饲料喂养联合腹腔注射链脲佐菌素建立糖尿病动脉粥样硬化小鼠模型，随机分为模型组、TZQ中、高剂量干预组，每组12只，每日分别给予0.9%氯化钠溶液10 mL/（kg·d）、中剂量TZQ 4.23 g/（kg·d）、高剂量TZQ 8.46 g/（kg·d）灌胃。另设12只雄性ApoE^-/-小鼠喂养普通饲料作为对照组，连续10周。实验结束后，取血检测小鼠血糖、血脂4项水平；油红O染色和苏木精-伊红（HE）染色法观察小鼠主动脉粥样硬化病变；酶联免疫吸附测定（ELISA）法检测血清炎症因子C-反应蛋白（CRP）、肿瘤坏死因子-α（TNF-α）和干扰素γ（IFN-γ）水平；实时荧光定量聚合酶链反应（qRT-PCR）和蛋白免疫印迹（Western blot）法测定主动脉组织中胞葬骨架重排相关因子Ras同系物家族成员A（RhoA），Rho相关蛋白激酶1、2（ROCK1、ROCK2）mRNA水平及蛋白表达水平。[结果] 与对照组相比，模型组小鼠出现精神萎靡、形体消瘦、气喘、行动减少、毛色黄、耳鼻唇舌颜色淡白、大便黏滞松软等表现，HE染色及油红O染色显示主动脉斑块面积增加，随机血糖与血清三酰甘油（TG）、总胆固醇（TC）、低密度脂蛋白胆固醇（LDL-C） 、高密度脂蛋白胆固醇（HDL-C）水平升高，血清炎症因子CRP、TNF-α、IFNγ水平升高，胞葬细胞骨架重排调节因子RhoA、ROCK1、ROCK2的 mRNA及蛋白表达水平升高（P<0.05或P<0.01），胞葬作用受损；与模型组比较，糖脂清中、高剂量干预组小鼠一般情况有所改善、主动脉斑块面积减小，随机血糖与血清TC、TG 和 LDL-C水平降低，血清炎症因子CRP、TNF-α、IFNγ水平降低，胞葬细胞骨架重排调节因子RhoA、ROCK1、ROCK2的mRNA水平及蛋白表达降低（P<0.05或P<0.01），胞葬作用改善；与中剂量组比较，高剂量组在减小主动脉斑块面积，降血脂及降低血清炎症因子水平，改善胞葬作用方面更具有优势（P<0.05或P<0.01）。[结论] TZQ可能通过降低抑制细胞骨架重排相关因子RhoA，ROCK1和ROCK2的表达，改善胞葬作用，从而发挥抗糖尿病动脉粥样硬化的作用。

Abstract:

[Objective] To explore the mechanism of Tangzhiqing Formula（TZQ） in treating diabetic atherosclerosis. [Methods] Thirty-six male ApoE^-/- mice were fed with high-fat diet combined with intraperitoneal injection of streptozotocin to establish the diabetic atherosclerosis model. They were randomly divided into model group，Tangzhiqing Formula medium and high dose intervention groups，with 12 mice in each group. 0.9% sodium chloride solution 10 mL/（kg·d），medium dose Tangzhiqing Formula 4.23 g/（kg·d） and high dose Tangzhiqing Formula 8.46 g/（kg·d） were administered by gavage each day. Another 12 male ApoE^-/- mice were fed with regular feed as the control group for 10 consecutive weeks. After the experiment，blood samples were taken to test the levels of blood glucose and blood lipids in mice；oil red O staining and HE staining were used to observe aortic atherosclerosis in mice；ELISA method was used to detect the levels of serum inflammatory factors C-reactive protein（CRP），tumor necrosis factor-α（TNF-α），and interferon gamma（IFN-γ）；QRT PCR and Western blot were used to measure the mRNA and protein expression levels of Ras homolog family member A（RhoA），Rho associated protein kinases 1 and 2（ROCK1，ROCK2），and other factors related to cytoskeletal rearrangement in aortic tissue. [Results] Compared with the control group，the model group mice showed symptoms such as mental fatigue，emaciation，wheezing，reduced mobility，yellow fur color，pale color of ears，nose，lips，and tongue，and loose and sticky stool. HE staining and oil red O staining showed an increase in aortic plaque area，random blood glucose and serum triglyceride（TG），total cholesterol（TC），low-density lipoprotein cholesterol（LDL-C），high-density lipoprotein cholesterol（HDL-C） levels increased，serum inflammatory factors CRP，TNF-α，IFN-γ levels increased，and mRNA and protein expression levels of cytoskeleton rearrangement related factors RhoA，ROCK1，and ROCK2 increased（P<0.05 or P<0.01），indicating impaired efferocytosis function；compared with the model group，the mice in the medium and high-dose intervention groups of Tangzhiqing Formula showed general improvement，reduced aortic plaque area，decreased random blood glucose and serum TC，TG，and LDL-C levels，decreased serum inflammatory factors CRP，TNF-α，and IFN-γ levels，and decreased mRNA and protein expression of cytoskeleton rearrangement related factors RhoA，ROCK1，and ROCK2（P<0.05 or P<0.01），indicating improved efferocytosis effects；compared with the medium dose group，the high-dose group has more advantages in reducing aortic plaque area，lowering blood lipids and serum inflammatory factor levels，and improving efferocytosis effects（P<0.05 or P<0.01）. [Conclusion] Tangzhiqing Formula may play an anti diabetic atherosclerosis role by down regulating the expression of cytoskeleton rearrangement related factors RhoA，ROCK1 and ROCK2，and improving the efferocytosis effect.