磷脂酸通过LPAR1/Akt途径诱导黄韧带肥大的损伤机制研究

Study on the injury mechanism of phosphatidic acid inducing ligamentum flavum hypertrophy through LPAR1/Akt pathway

目的探究磷脂酸通过溶血磷脂酸受体1(LPAR1)/丝苏氨酸蛋白激酶(Akt)途径诱导黄韧带肥大的损伤机制研究。方法回顾性选择2019年10月至2020年3月于宝鸡市中医医院接受腰椎管狭窄症减压椎板切除术30例患者的黄韧带组织样本为观察组,另选同期接受了类似手术的无腰椎管狭窄症的30例患者作为对照组,免疫组化分析组织样本中LPA和LPAR1的表达,蛋白印迹分析胶原蛋白Ⅰ和胶原蛋白Ⅲ的表达。将黄韧带细胞分为3组,即黄韧带细胞组(不进行任何处理)、LPA处理组(10μmol/L的LPA)和LPAR1抑制组(10μmol/L的LPA+终浓度50μmol/L的LPAR1抑制剂),干预12 h和36 h后,细胞计数试剂盒-8(CCK-8)检测细胞的活力及增殖;干预24 h后,膜联蛋白V-APC和碘化丙啶染色并使用流式细胞仪检测细胞凋亡率;实时聚合酶链式反应(RT-PCR)分析各组细胞中Caspase 3、Bax、Bcl-2的mRNA表达;蛋白印迹法分析Akt、Cdk1和Cyclin-B1蛋白表达。结果观察组较对照组LPA和LPAR1的表达升高(P<0.05)。观察组较对照组Ⅰ型胶原和Ⅲ型胶原的蛋白表达升高(P<0.05)。LPA处理组较黄韧带细胞组细胞增殖升高(P<0.05),LPAR1抑制剂组较LPA处理组细胞增殖降低(P<0.05)。LPA处理组较黄韧带细胞组细胞活力升高(P<0.05),LPAR1抑制剂较LPA处理组细胞活力降低(P<0.05),LPA处理组较黄韧带细胞组细胞凋亡降低(P<0.05)。LPA处理组较黄韧带细胞组Caspase 3、Bax、Bcl-2的mRNA表达降低(P<0.05),LPAR1抑制剂较磷脂酸处理组Caspase 3、Bax、Bcl-2的mRNA表达升高(P<0.05)。LPA处理组较黄韧带细胞组P-Akt、Cdk、Cyclin的表达升高(P<0.05),LPAR1抑制剂组较LPA处理组P-Akt、Cdk、Cyclin的表达降低(P<0.05)。结论LPA-LPAR1-Akt的激活与黄韧带细胞的增殖和存活密切相关,LPAR抑制剂对磷脂酸化黄韧带细胞的细胞活力和增殖具有抑制作用,并通过促进凋亡相关蛋白的表达和抑制磷酸化途径蛋白的表达对黄韧带肥大损伤发挥治疗作用。

Objective To explore the mechanism of phosphatidic acid inducing ligamentum flavum hypertrophy through the LPAR1/Akt pathway.Methods The tissue samples of the ligamentum flavum were collected from 30 patients undergoing lumbar spinal stenosis decompression laminectomy at Baoji Traditional Chinese Medicine Hospital from October 2019 to March 2020 as the observation group.Another 30 patients without lumbar spinal stenosis who underwent similar surgery during the same period were selected as the control group.Immunohistochemical analysis of the expression of LPA and LPAR1 in tissue samples,Western blotting analysis of the expression of collagenⅠand collagenⅢ.The ligamentum flavum cells erre divided into three groups,namely ligamentum flavum cell group(without any treatment),LPA treatment group(10μmol/L LPA)and LPAR1 inhibition group(10μmol/L LPA+final concentration of 50μmol/L LPAR1 inhibitor),after intervention for 12 h and 36 h,cell counting kit-8(CCK-8)detects cell viability and proliferation;after 24 hours of intervention,annexin V-APC and propidium iodide(Annexin V-FITC/PI)were stained and the apoptosis rate was detected by flow cytometry;RT-PCR real-time analysis of the mRNA expression of Caspase 3,Bax and Bcl-2 in each group of cells;Western blot analysis of Akt,Cdk1 and Cyclin-B1 protein expression.Results The expression of LPA and LPAR1 in the observation group was higher than that in the control group(P<0.05).The protein expression of typeⅠcollagen and typeⅢcollagen in the observation group was higher than that in the control group(P<0.05).The cell proliferation of the phosphatidic acid treatment group was higher than that of the ligamentum flavum cell group(P<0.05),and the cell proliferation of the LPAR1 inhibitor was lower than that of the phosphatidic acid treatment group(P<0.05).The cell viability of the phosphatidic acid treatment group was higher than that of the ligamentum flavum cell group(P<0.05),the cell viability of LPAR1 inhibitor was lower than that of the phosphatidic acid treatment group(P<0.05),and the apoptosis of the phosphatidic acid treatment group was lower than that of the ligamentum flavum cell group(P<0.05).The mRNA expression of Caspase 3,Bax and Bcl-2 in the phosphatidic acid treatment group was lower than that in the ligamentum flavum cell group(P<0.05),and the mRNA expression of Caspase 3,Bax and Bcl-2 in the LPAR1 inhibitor was higher than that in the phosphatidic acid treatment group(P<0.05).The expression of P-Akt,Cdk and Cyclin in the phosphatidic acid treatment group was higher than that in the ligamentum flavum cell group(P<0.05),and the expression of P-Akt,Cdk and Cyclin in the LPAR1 inhibitor group was lower than that in the phosphatidic acid treatment group(P<0.05).Conclusion The activation of LPA-LPAR1-Akt is closely related to the proliferation and survival of ligamentum flavum cells.LPAR inhibitors can inhibit the cell viability and proliferation of phosphatidic acidified ligamentum flavum cells,and it has a therapeutic effect on ligamentum flavum hypertrophy injury by promoting the expression of apoptosis-related proteins and inhibiting the expression of phosphorylation pathway proteins.