基于TRPA1调控泡沫细胞胆固醇代谢作用探讨枳实薤白桂枝汤治疗AS的作用机制

Mechanism of Zhishi Xiebai Guizhitang in Treating AS Based on Regulation of Cholesterol Metabolism in Foam Cells by TRPA1

目的:基于主动脉瞬时受体电位通道锚蛋白1(TRPA1)调控泡沫细胞胆固醇代谢作用探讨枳实薤白桂枝汤对动脉粥样硬化(AS)小鼠疾病进展的影响与作用机制。方法:采用高脂饲料在载脂蛋白E敲除(ApoE^(-/-))小鼠上建立AS模型,随机分为枳实薤白桂枝汤低、中、高剂量组(2.97、5.94、11.88 g·kg^(-1))与辛伐他汀组(0.002 g·kg^(-1)),每组织喂食高脂饲料同时给药,C57BL/6J小鼠作为正常组使用普通饲料喂养,每组10只。上述过程结束后,取小鼠主动脉与血清。采用苏木素-伊红(HE)染色法观察主动脉根部病理变化;大体油红观察主动脉脂质斑块情况;检测血清中总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)水平,并用酶联免疫吸附测定法(ELISA)检测白细胞介素-1β(IL-1β)、IL-18水平;蛋白免疫印迹法(Western blot)与免疫组化分析TRPA1、三磷酸腺苷(ATP)结合盒转运体A1(ABCA1)、ATP结合盒转运体G1(ABCG1)及甘露糖受体(CD206)表达情况。结果:从药效上看,与正常组比较,模型组主动脉出现斑块、大量泡沫细胞与胆固醇晶体等病理变化,血清TC、LDL-C、IL-1β、IL-18水平均显著升高(P<0.01),HDL-C水平显著下降(P<0.01),主动脉组织CD206水平显著下降(P<0.01);与模型组比较,各给药组均能改善主动脉上的脂质沉积状况,并且除枳实薤白桂枝汤高剂量组外,各给药组均能显著降低TC、LDL-C水平(P<0.01),炎症方面,除枳实薤白桂枝汤中剂量组外,各给药组的IL-1β、IL-18水平均明显降低(P<0.05),此外,枳实薤白桂枝汤可上调CD206水平,且枳实薤白桂枝汤中、高剂量组差异明显(P<0.05)。从作用机制上看,与正常组比较,模型组的主动脉TRPA1、ABCA1、ABCG1表达水平均明显降低(P<0.05);与模型组比较,各给药组均明显提高了主动脉TRPA1表达量(P<0.05),可提高ABCA1、ABCG1的表达量,其中枳实薤白桂枝汤中、高剂量组和辛伐他汀组差异明显(P<0.05),免疫组化结果的趋势与其基本一致。结论:枳实薤白桂枝汤可有效降低血脂与炎症水平,抑制主动脉斑块的形成,其作用机制可能是通过TRPA1引发下游的ABCA1、ABCG1表达升高,促进泡沫细胞中胆固醇流出,从而调控胆固醇代谢并在一定程度上干预炎症水平,最终达到治疗AS的作用。

Objective:To explore the effect and mechanism of Zhishi Xiebai Guizhitang on the progression of atherosclerosis(AS)mice based on the regulation of cholesterol metabolism in foam cells by transient receptor potential channel ankyrin 1(TRPA1).Method:The AS model was established on apolipoprotein E knockout(ApoE^(-/-))mice with a high-fat diet.The mice were randomly divided into low-dose,middle-dose,and high-dose groups of Zhishi Xiebai Guizhitang(2.97,5.94,11.88 g·kg^(-1))and simvastatin group(0.002 g·kg^(-1)),and the drug was administered along with a high-fat diet.C57BL/6J mice were fed an ordinary diet as a normal group.After the above process,the aorta and serum of mice were taken.The pathological changes of the aortic root were observed by hematoxylin-eosin(HE)staining.The lipid plaques in the aorta were observed by gross oil redness.Serum levels of total cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol(LDL-C),and high density lipoprotein cholesterol(HDL-C)were detected,and the levels of interleukin-1β(IL-1β)and interleukin-18(IL-18)were detected by enzyme-linked immunosorbent assay(ELISA).Western blot and immunohistochemical method were used to analyze the expression of TRPA1,ATP-binding cassette transporter A1(ABCA1),ATP-binding cassette transporter G1(ABCG1),and mannose receptor(CD206).Result:From the perspective of drug efficacy,compared with the normal group,pathological changes such as plaque,a large number of foam cells,and cholesterol crystals appeared in the aorta of the model group,and the serum levels of TC,LDL-C,IL-1β,and IL-18 were significantly increased(P<0.01).The HDL-C level was significantly decreased(P<0.01),and the CD206 level in aortic tissue was significantly decreased(P<0.01).Compared with the model group,the lipid deposition in the aorta was alleviated in all drug administration groups.In addition,except for the high-dose group of Zhishi Xiebai Guizhitang,all drug administration groups could significantly decrease the levels of TC and LDL-C(P<0.01).In terms of inflammation,except for the middle-dose group of Zhishi Xiebai Guizhitang,the levels of IL-1β and IL-18 were significantly decreased in all drug administration groups(P<0.05).Moreover,Zhishi Xiebai Guizhitang could also up-regulate the levels of CD206,and the difference was significant in the middle-dose and high-dose groups(P<0.05).From the perspective of mechanism,the expression levels of TRPA1,ABCA1,and ABCG1 in the aorta in the model group were lower than those in the normal group(P<0.05).Compared with the model group,all drug administration groups significantly increased the expression of TRPA1 in the aorta(P<0.05),and the expressions of ABCA1 and ABCG1 were increased.The differences in the middle-dose and highdose groups and the simvastatin group were significant(P<0.05),which was basically consistent with the trend of immunohistochemical results.Conclusion:Zhishi Xiebai Guizhitang can effectively reduce blood lipid and inflammation levels and inhibit the formation of aortic plaque.The mechanism may be explained as follows:the expressions of ABCA1 and ABCG1 downstream are increased through TRPA1,which promotes cholesterol outflow in foam cells,thereby regulating cholesterol metabolism,intervening in inflammation level to a certain extent,and finally treating AS.