香砂六君子汤对脾虚高脂血症大鼠dyHDL的影响
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摘要
目的:观察脾虚高脂血症大鼠失功能高密度脂蛋白胆固醇(dyHDL)变化及香砂六君子汤的干预作用,从HDL"质"的变化,揭示香砂六君子汤对脾虚高脂血症大鼠dy HDL的影响及机制。方法:75只SPF级SD大鼠随机分为正常组,高脂组,脾虚高脂组,香砂六君子低、高剂量组(5. 67,11. 34 g·kg-1),脾虚高脂组,香砂六君子低、高剂量组采用饮食不节加力竭游泳的复合方法造模15 d后,正常组喂饲基础饲料,高脂组,脾虚高脂组,香砂六君子、高剂量组喂饲高脂饲料。12周后,香砂六君子低、高剂量组给予相对剂量药物,正常组、高脂组、脾虚高脂组给予相应体积生理盐水。4周后,全自动生化分析仪检测血清总胆固醇(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)含量,间苯三酚法检测大鼠D-木糖排泄率,苏木素-伊红(HE)染色观察肝脏细胞形态变化,酶联免疫吸附测定(ELISA)检测血浆对氧磷酶1(PON1),载脂蛋白A1(apo A1),血清淀粉样蛋白A(SAA)含量,实时荧光定量PCR(Real-time PCR)检测大鼠肝脏PON1,apo A1,SAA mRNA表达水平。结果:与正常组比较,高脂组及脾虚高脂组大鼠血清TC,TG,LDL-C水平明显升高(P <0. 05),HDL-C水平明显降低(P <0. 05),高脂组及脾虚高脂组大鼠血浆PON1,apo A1含量明显降低(P <0. 05),SAA含量明显升高(P <0. 05),肝脏组织SAA mRNA显著升高(P <0. 01),PON1,apo A1 mRNA表达显著降低(P <0. 01),高脂组及脾虚高脂组大鼠肝细胞肿大变圆、可见散在的脂肪空泡,且脾虚高脂组大鼠尿D-木糖排泄率明显降低(P <0. 05)。与高脂组及脾虚高脂组比较,香砂六君子汤低、高剂量组血清TC,LDL-C水平明显降低(P <0. 05); HDL-C水平明显升高(P <0. 05),血浆PON1,apo A1含量明显升高(P <0. 05),SAA含量明显降低(P <0. 05),肝脏组织SAA mRNA明显降低(P <0. 05),apo A1 mRNA表达明显升高(P <0. 05),肝脏细胞肿胀明显减轻、脂肪泡沫化减少。与高脂组相比,脾虚高脂组大鼠血浆中PON1,SAA含量明显降低(P <0. 05),肝细胞肿胀明显、泡沫化加重。结论:脾虚状态下高脂血症大鼠脂质紊乱加重,香砂六君子汤干预后紊乱得以纠正,其作用机制可能与调控dy HDL相关mRNA及蛋白表达有关。
Objective: To observe the changes of dysfunctional high density lipoprotein cholesterol( dyHDL) and the intervention effect of Xiangsha Liu Junzitang in rats with spleen deficiency and hyperlipidemia,and reveal the effect and mechanism of Xiangsha Liu Junzitang on dyHDL in rats with spleen-deficiency hyperlipidemia. Method: Seventy-five SPF SD rats were randomly divided into normal group,high fat group,spleen deficiency and high fat group,Xiangsha Liu Junzitang low and high dose groups( 5. 67,11. 34 g·kg-1).In the spleen deficiency and high fat group,as well as Xiangsha Liu Junzitang low and high dose groups,composite method of improper diet and exhaustive swimming was used for 15 days for modeling. After modeling for 15 days,normal group was fed with basic diet,while the high-fat group,spleen-deficiency and high-fat group,the Xiangsha Liu Junzitang low and high dose groups were fed with high-fat diet. After 12 weeks,the Xiangsha Liu Junzitang low dose and high dose groups received corresponding dosage of drugs,while normal group,high fat group and spleen deficiency high fat group received corresponding volume of normal saline. After 4 weeks,the contents of total cholesterol( TC),triglyceride( TG),low density lipoprotein cholesterol cholesterol( LDL-C),and high density lipoprotein cholesterol( HDL-C) were detected by automatic biochemistry analyzer,while D-xylose excretion rate was measured by phloroglucinol method. The morphological changes of liver cells were observed by hematoxylin eosin( HE) staining. The level of PON1, apoA1 and SAA in plasma were detected by enzyme-linked immunosorbent assay( ELISA). Paraoxonase 1( PON1),apolipoprotein A1( apoA1) and serum amyloid protein A( SAA) gene expression in rats liver were detected by Real-time fluorescence quantitative polymerase chain reaction( Real-time PCR). Result: As compared with normal group,the serum TC,TG,and LDL-C levels were significantly increased in the high-fat group and spleen-deficiency high-fat group( P < 0. 05),and HDL-C levels were significantly lower( P < 0. 05); the levels of PON1 and apoA1 in plasma were decreased( P < 0. 05),while the content of SAA was increased( P < 0. 05); the expression of SAA gene in liver tissues was increased( P <0. 01),and the expression levels of PON1 and apoA1 genes in liver tissues were significantly decreased( P <0. 01). In high-fat group and the spleen-deficiency and high-fat group, the hepatocytes were rounded and scattered,and scattered fat vacuoles were observed. In addition, the urinary D-xylose excretion rate was significantly decreased in the spleen-deficiency and high-fat group( P < 0. 05). After the intervention with Xiangsha Liu Junzitang,the serum TC,LDL-C levels were significantly decreased( P < 0. 05); HDL-C levels were significantly increased( P < 0. 05); plasma PON1,apoA1 levels were increased( P < 0. 05); the content of SAA was decreased( P < 0. 05); the expression of SAA gene in liver tissues was decreased( P < 0. 05); the expression of apoA1 gene was increased significantly( P < 0. 05). Liver cells swelling was significantly alleviated and fat foaming was reduced. As compared with high-fat group,the plasma PON1 and SAA levels in the spleen-deficiency high-fat group were significantly lower( P < 0. 05); hepatocyte swelling was obvious and foaming was aggravated.Conclusion: The lipid disorder in hyperlipidemia rats was aggravated by the spleen deficiency,but was corrected after intervention with Xiangsha Liu Junzitang. and its mechanism may be related to the regulation of the expression of dyHDL-related genes and protein.
Objective: To observe the changes of dysfunctional high density lipoprotein cholesterol( dyHDL) and the intervention effect of Xiangsha Liu Junzitang in rats with spleen deficiency and hyperlipidemia,and reveal the effect and mechanism of Xiangsha Liu Junzitang on dyHDL in rats with spleen-deficiency hyperlipidemia. Method: Seventy-five SPF SD rats were randomly divided into normal group,high fat group,spleen deficiency and high fat group,Xiangsha Liu Junzitang low and high dose groups( 5. 67,11. 34 g·kg-1).In the spleen deficiency and high fat group,as well as Xiangsha Liu Junzitang low and high dose groups,composite method of improper diet and exhaustive swimming was used for 15 days for modeling. After modeling for 15 days,normal group was fed with basic diet,while the high-fat group,spleen-deficiency and high-fat group,the Xiangsha Liu Junzitang low and high dose groups were fed with high-fat diet. After 12 weeks,the Xiangsha Liu Junzitang low dose and high dose groups received corresponding dosage of drugs,while normal group,high fat group and spleen deficiency high fat group received corresponding volume of normal saline. After 4 weeks,the contents of total cholesterol( TC),triglyceride( TG),low density lipoprotein cholesterol cholesterol( LDL-C),and high density lipoprotein cholesterol( HDL-C) were detected by automatic biochemistry analyzer,while D-xylose excretion rate was measured by phloroglucinol method. The morphological changes of liver cells were observed by hematoxylin eosin( HE) staining. The level of PON1, apoA1 and SAA in plasma were detected by enzyme-linked immunosorbent assay( ELISA). Paraoxonase 1( PON1),apolipoprotein A1( apoA1) and serum amyloid protein A( SAA) gene expression in rats liver were detected by Real-time fluorescence quantitative polymerase chain reaction( Real-time PCR). Result: As compared with normal group,the serum TC,TG,and LDL-C levels were significantly increased in the high-fat group and spleen-deficiency high-fat group( P < 0. 05),and HDL-C levels were significantly lower( P < 0. 05); the levels of PON1 and apoA1 in plasma were decreased( P < 0. 05),while the content of SAA was increased( P < 0. 05); the expression of SAA gene in liver tissues was increased( P <0. 01),and the expression levels of PON1 and apoA1 genes in liver tissues were significantly decreased( P <0. 01). In high-fat group and the spleen-deficiency and high-fat group, the hepatocytes were rounded and scattered,and scattered fat vacuoles were observed. In addition, the urinary D-xylose excretion rate was significantly decreased in the spleen-deficiency and high-fat group( P < 0. 05). After the intervention with Xiangsha Liu Junzitang,the serum TC,LDL-C levels were significantly decreased( P < 0. 05); HDL-C levels were significantly increased( P < 0. 05); plasma PON1,apoA1 levels were increased( P < 0. 05); the content of SAA was decreased( P < 0. 05); the expression of SAA gene in liver tissues was decreased( P < 0. 05); the expression of apoA1 gene was increased significantly( P < 0. 05). Liver cells swelling was significantly alleviated and fat foaming was reduced. As compared with high-fat group,the plasma PON1 and SAA levels in the spleen-deficiency high-fat group were significantly lower( P < 0. 05); hepatocyte swelling was obvious and foaming was aggravated.Conclusion: The lipid disorder in hyperlipidemia rats was aggravated by the spleen deficiency,but was corrected after intervention with Xiangsha Liu Junzitang. and its mechanism may be related to the regulation of the expression of dyHDL-related genes and protein.
引文
[1]朱美林,贾连群,杨关林,等.脾虚状态对高脂血症大鼠肝脏胆固醇代谢的影响及机制研究[J].中华中医药杂志,2015,30(8):2712-2716.
[2]黄珮,郭姣,朴胜华.高脂血症中医证候实质研究进展[J].辽宁中医杂志,2011,38(9):1904-1907.
[3]李维娜,冯玲.高脂血症从脾论治探幽[J].世界中西医结合杂志,2017,12(4):577-580.
[4]朱美林.脾虚状态对高脂血症大鼠肝脏脂质沉积的影响及机制研究[D].沈阳:辽宁中医药大学,2016.
[5]吕美君,贾连群,王志丹,等.香砂六君子汤的文献分析研究[J].中华中医药学刊,2016,34(7):1620-1623.
[6]奚锦要,朱永钦,朱永苹,等.香砂六君子汤联合针刺疗法治疗功能性消化不良临床疗效观察[J].中国实验方剂学杂志,2017,23(20):157-162.
[7]黎豫川,贾波,彭腾,等.香砂六君子汤水煎液乙酸乙酯部位化学成分研究[J].中国实验方剂学杂志,2013,19(20):112-114.
[8]尔璐,边云飞,宋晓苏,等.失功能高密度脂蛋白与心血管疾病研究进展[J].中国动脉硬化杂志,2017,25(3):309-313.
[9]王静,林晶晶,沈涛.高脂血症的现代医学研究[J].山西医药杂志,2015,44(19):2243-2245.
[10]郑小伟.中医实验动物模型方法学[M].上海:上海中医学院出版社,1999:63-111.
[11]姜晓琳,张立德.脾气虚证模型大鼠中BNP介导的c AMP-PKA信号通路对b FGF表达的影响[J].中国病理生理杂志,2016,32(2):251-255.
[12]陈雷,李海权.中医脾虚大鼠证候动物模型评价系统软件研究[J].中华中医药学刊,2010,28(2):370-371.
[13]王晓明,易杰,廖世新,等.脾虚证动物模型的客观评估[J].中华中医药杂志,2006,21(7):406-408.
[14]黄永艳.化痰活血方对高脂血症大鼠调脂作用的机制研究[D].武汉:湖北中医学院,2006.
[15]陈奇.中药药理实验方法[M].北京:人民卫生出版社,1994:205-208.
[16]贾连群,杨关林,张哲,等.从“脾主运化”理论探讨膏脂转输与胆固醇逆向转运[J].中医杂志,2013,54(20):1793-1795.
[17]林巧云.周仲瑛教授从“痰瘀”辨治高脂血症的临床经验及学术思想研究[D].南京:南京中医药大学,2017.
[18]杨胜兰.高脂血症的基本病机探讨[J].中医杂志,2005,46(11):861-863.
[19]贾连群,宋囡,张妮,等.基于“脾主运化”理论探讨肠道微生物稳态与膏脂转输的关系[J].中医杂志,2017,58(18):1554-1557.
[20]李娜,龙石银,陈志军,等.代谢综合征患者血浆甘油三酯水平对HDL亚类分布的影响[J].中国动脉硬化杂志,2015,23(3):285-289.
[21]翟振丽,马维红,李全忠,等.高密度脂蛋白功能差异与动脉粥样硬化的研究进展[J].实用医学杂志,2013,29(21):3608-3609.
[22]尔璐,边云飞,宋晓苏,等.失功能高密度脂蛋白与心血管疾病研究进展[J].中国动脉硬化杂志,2017,25(3):309-313.
[23]应如,秦亚飞,郭志刚,等.失功能高密度脂蛋白致动脉粥样硬化的研究进展[J].广东医学,2012,33(11):1671-1673.
[24]闫丽荣.冠心病患者血浆HDL组分中apo C-Ⅰ,SAA水平变化及SAA mRNA多态性研究[D].北京:北京协和医学院,2014.
[25]王子熹.吸烟对高密度脂蛋白功能的影响[D].北京:北京协和医学院,2009.
[26]田迪,秦亚飞,应如,等.辛伐他汀对Apo E-/-小鼠HDL抗炎抗氧化功能的影响[J].解放军医学杂志,2013,38(3):195-200.
[27] Bounafaa A,Berrougui H,Ikhlef S,et al. Alteration of HDL functionality and PON1 activities in acute coronary syndrome patients[J]. Clin Biochem,2014,47(18):318-325.
[28]曾宪钦,胡雪松.血清PON1活性与冠状动脉粥样硬化研究新进展[J].中国实用医药,2008,3(12):179-181.
[29]黄辰,李瑛,赵妍,等.脾虚证动物模型评价方法评述[J].辽宁中医杂志,2018,45(2):433-437,447.
[2]黄珮,郭姣,朴胜华.高脂血症中医证候实质研究进展[J].辽宁中医杂志,2011,38(9):1904-1907.
[3]李维娜,冯玲.高脂血症从脾论治探幽[J].世界中西医结合杂志,2017,12(4):577-580.
[4]朱美林.脾虚状态对高脂血症大鼠肝脏脂质沉积的影响及机制研究[D].沈阳:辽宁中医药大学,2016.
[5]吕美君,贾连群,王志丹,等.香砂六君子汤的文献分析研究[J].中华中医药学刊,2016,34(7):1620-1623.
[6]奚锦要,朱永钦,朱永苹,等.香砂六君子汤联合针刺疗法治疗功能性消化不良临床疗效观察[J].中国实验方剂学杂志,2017,23(20):157-162.
[7]黎豫川,贾波,彭腾,等.香砂六君子汤水煎液乙酸乙酯部位化学成分研究[J].中国实验方剂学杂志,2013,19(20):112-114.
[8]尔璐,边云飞,宋晓苏,等.失功能高密度脂蛋白与心血管疾病研究进展[J].中国动脉硬化杂志,2017,25(3):309-313.
[9]王静,林晶晶,沈涛.高脂血症的现代医学研究[J].山西医药杂志,2015,44(19):2243-2245.
[10]郑小伟.中医实验动物模型方法学[M].上海:上海中医学院出版社,1999:63-111.
[11]姜晓琳,张立德.脾气虚证模型大鼠中BNP介导的c AMP-PKA信号通路对b FGF表达的影响[J].中国病理生理杂志,2016,32(2):251-255.
[12]陈雷,李海权.中医脾虚大鼠证候动物模型评价系统软件研究[J].中华中医药学刊,2010,28(2):370-371.
[13]王晓明,易杰,廖世新,等.脾虚证动物模型的客观评估[J].中华中医药杂志,2006,21(7):406-408.
[14]黄永艳.化痰活血方对高脂血症大鼠调脂作用的机制研究[D].武汉:湖北中医学院,2006.
[15]陈奇.中药药理实验方法[M].北京:人民卫生出版社,1994:205-208.
[16]贾连群,杨关林,张哲,等.从“脾主运化”理论探讨膏脂转输与胆固醇逆向转运[J].中医杂志,2013,54(20):1793-1795.
[17]林巧云.周仲瑛教授从“痰瘀”辨治高脂血症的临床经验及学术思想研究[D].南京:南京中医药大学,2017.
[18]杨胜兰.高脂血症的基本病机探讨[J].中医杂志,2005,46(11):861-863.
[19]贾连群,宋囡,张妮,等.基于“脾主运化”理论探讨肠道微生物稳态与膏脂转输的关系[J].中医杂志,2017,58(18):1554-1557.
[20]李娜,龙石银,陈志军,等.代谢综合征患者血浆甘油三酯水平对HDL亚类分布的影响[J].中国动脉硬化杂志,2015,23(3):285-289.
[21]翟振丽,马维红,李全忠,等.高密度脂蛋白功能差异与动脉粥样硬化的研究进展[J].实用医学杂志,2013,29(21):3608-3609.
[22]尔璐,边云飞,宋晓苏,等.失功能高密度脂蛋白与心血管疾病研究进展[J].中国动脉硬化杂志,2017,25(3):309-313.
[23]应如,秦亚飞,郭志刚,等.失功能高密度脂蛋白致动脉粥样硬化的研究进展[J].广东医学,2012,33(11):1671-1673.
[24]闫丽荣.冠心病患者血浆HDL组分中apo C-Ⅰ,SAA水平变化及SAA mRNA多态性研究[D].北京:北京协和医学院,2014.
[25]王子熹.吸烟对高密度脂蛋白功能的影响[D].北京:北京协和医学院,2009.
[26]田迪,秦亚飞,应如,等.辛伐他汀对Apo E-/-小鼠HDL抗炎抗氧化功能的影响[J].解放军医学杂志,2013,38(3):195-200.
[27] Bounafaa A,Berrougui H,Ikhlef S,et al. Alteration of HDL functionality and PON1 activities in acute coronary syndrome patients[J]. Clin Biochem,2014,47(18):318-325.
[28]曾宪钦,胡雪松.血清PON1活性与冠状动脉粥样硬化研究新进展[J].中国实用医药,2008,3(12):179-181.
[29]黄辰,李瑛,赵妍,等.脾虚证动物模型评价方法评述[J].辽宁中医杂志,2018,45(2):433-437,447.