摘要
目的基于河马(Hippo)-Yes相关蛋白(YAP)/带有PDZ结合基序的转录共激活因子(TAZ)信号通路探讨吴门枳壳甘草汤延缓椎间盘退变的可能作用机制。方法50只SD大鼠随机分为假手术组、模型组、枳壳甘草汤低剂量组、枳壳甘草汤高剂量组、枳壳甘草汤高剂量+抑制剂组,每组10只。假手术组大鼠采用21G针头在尾部Co6/7/8节段穿刺皮肤和肌肉(深度约为2 mm),不损伤椎间盘,其余各组大鼠在尾部Co6/7/8节段用21G针头穿刺尾椎间盘5 mm建立椎间盘退变大鼠模型。造模1周后,枳壳甘草汤低、高剂量组分别予6.24、12.24 g/(kg·d)枳壳甘草汤灌胃,枳壳甘草汤高剂量+抑制剂组大鼠给予12.24 g/(kg·d)枳壳甘草汤灌胃后,按10 mg/kg腹腔注射YAP/TAZ抑制剂Verteporfin,假手术组与模型组大鼠予5 ml/(kg·d)生理盐水灌胃。灌胃每日1次,腹腔注射隔日1次。各组均连续干预4周后,采用HE染色、番红O-固绿染色及甲苯胺蓝染色观察大鼠尾椎间盘病理改变;免疫组化法检测椎间盘髓核组织聚集蛋白聚糖(Aggrecan)、基质金属蛋白酶3(MMP3)表达;Tunel荧光染色检测髓核组织细胞凋亡,计算凋亡率;Western blot法检测髓核组织中Hippo-YAP/TAZ信号通路[包括YAP、磷酸化Yes相关蛋白(p-YAP)、磷酸化20样激酶1/2(p-MST1/2)、带有PDZ结合基序的磷酸化转录共激活因子(p-TAZ)]及凋亡相关蛋白[包括剪切的含半胱氨酸的天冬氨酸蛋白水解酶(Cleaved Caspase 3)、肿瘤基因P53(P53)、B淋巴细胞瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)]水平。结果与假手术组比较,模型组大鼠椎间盘发生明显退行性改变,椎间盘髓核组织Aggrecan、Bcl-2、YAP蛋白水平降低,p-MST1/2、p-YAP、p-TAZ、P53、Bax、Cleaved Caspase 3、MMP3蛋白水平及细胞凋亡率均上升(P<0.01)。与模型组比较,各药物干预组椎间盘退变均得到一定恢复,Aggrecan、Bcl-2、YAP蛋白水平均升高,p-MST1/2、p-YAP、p-TAZ、P53、Bax、Cleaved Caspase 3、MMP3蛋白水平及细胞凋亡率均降低(P<0.05或P<0.01)。与枳壳甘草汤低剂量组比较,枳壳甘草汤高剂量组大鼠椎间盘退变恢复更显著,p-MST1/2、p-YAP、p-TAZ、P53、Bax、Cleaved Caspase 3、MMP3蛋白水平及细胞凋亡率降低,Aggrecan、Bcl-2、YAP蛋白水平升高(P<0.05或P<0.01)。与枳壳甘草汤高剂量组比较,枳壳甘草汤高剂量+抑制剂组大鼠椎间盘退变恢复程度降低,p-MST1/2、p-YAP、p-TAZ、P53、Bax、Cleaved Caspase 3、MMP3蛋白水平及细胞凋亡率升高,Aggrecan、Bcl-2、YAP蛋白水平降低(P<0.05或P<0.01)。结论枳壳甘草汤可能通过抑制椎间盘髓核组织YAP磷酸化,维持Hippo-YAP/TAZ信号通路功能,降低髓核细胞凋亡,从而延缓椎间盘退变。
Objective To investigate the possible mechanism by which Zhiqiao Gancao Decoction(枳壳甘草汤,ZGD)delays intervertebral disc degeneration(IDD)based on the Hippo-yes-associated protein(YAP)/transcriptional co-activator with PDZ-binding motif(TAZ)signaling pathway.Methods A total of 50 SD rats were randomly divided into sham surgery group,model group,low-dose ZGD group,high-dose ZGD group,and high-dose ZGD+inhibitor group,with 10 rats in each group.In the sham surgery group,the rats were pierced in the skin and muscle at the Co6/7/8 segments of the tail with a 21G needle(depth approximately 2 mm)without damaging the intervertebral disc.In the other groups,rats were injected with a 21G needle at the Co6/7/8 segments of the tail to establish an IDD model by piercing the tail intervertebral disc 5 mm.One week after modeling,rats in the low-dose and high-dose ZGD groups were given 6.24 and 12.24 g/(kg·d)of the decoction via gastric gavage,respectively.The high-dose ZGD+inhibitor group was given 12.24 g/(kg·d)of the decoction and an intraperitoneal injection of YAP/TAZ inhibitor Verteporfin 10 mg/kg.The sham surgery and model groups were given 5 ml/(kg·d)of normal saline via gavage.The gavage was given once a day,and the intraperitoneal injection was given every other day.After 4 weeks of continuous intervention,the pathological changes of the tail intervertebral discs were observed using HE staining,Oil Red O-Green staining,and Toluidine Blue staining.Immunohistochemistry was used to detect the expression of aggrecan and MMP3 in the nucleus pulposus.TUNEL fluorescence staining was performed to detect apoptosis in the nucleus pulposus,and the apoptosis rate was calculated.Western blot was used to detect the Hippo-YAP/TAZ signaling pathway,including YAP,phosphorylated YAP(p-YAP),phosphorylated MST1/2(p-MST1/2),phosphorylated TAZ(p-TAZ)and apoptosis-related proteins,such as Cleaved Caspase 3,P53,Bcl-2 and Bax.Results Compared with sham surgery group,the rats in the model group showed significant degenerative changes in the intervertebral disc.The levels of aggrecan,Bcl-2,and YAP proteins in the nucleus pulposus decreased,while the levels of p-MST1/2,p-YAP,p-TAZ,P53,Bax,Cleaved Caspase 3,MMP3 proteins,and the apoptosis rate increased(P<0.01).Compared with the model group,the drug intervention groups showed partial recovery in intervertebral disc degeneration.The levels of aggrecan,Bcl-2,and YAP proteins increased,while the levels of p-MST1/2,p-YAP,p-TAZ,P53,Bax,Cleaved Caspase 3,MMP3 proteins,and the apoptosis rate decreased(P<0.05 or P<0.01).The high-dose ZGD group showed more significant recovery in intervertebral disc degeneration compared to the low-dose ZGD group,with a decrease in the levels of p-MST1/2,p-YAP,p-TAZ,P53,Bax,Cleaved Caspase 3,MMP3 proteins,and apoptosis rate,and an increase in the levels of aggrecan,Bcl-2,and YAP proteins(P<0.05 or P<0.01).Compared with the high-dose ZGD group,the high-dose ZGD+inhibitor group showed a reduced recovery in intervertebral disc degeneration,with an increase in the levels of p-MST1/2,p-YAP,p-TAZ,P53,Bax,Cleaved Caspase 3,MMP3 proteins,and apoptosis rate,and a decrease in the levels of aggrecan,Bcl-2,and YAP proteins(P<0.05 or P<0.01).Conclusion ZGD may delay intervertebral disc degeneration by inhibiting the phosphorylation of YAP in the nucleus pulposus,maintaining the function of the Hippo-YAP/TAZ signaling pathway,and reducing apoptosis of nucleus pulposus cells.
作者
朱在师
黄泽灵
陈俊名
徐波
陆斌杰
陈华
段星星
李宇卫
沈晓峰
ZHU Zaishi;HUANG Zeling;CHEN Junming;XU Bo;LU Binjie;CHEN Hua;DUAN Xingxing;LI Yuwei;SHEN Xiaofeng(Suzhou Hospital of Traditional Chinese Medicine Affiliated to Nanjing University of Chinese Medicine,Suzhou,215009;Orthopaedic and Traumatology Institute,Suzhou Academy of Chinese Medicine WU Sect)
出处
《中医杂志》
北大核心
2025年第5期509-517,共9页
Journal of Traditional Chinese Medicine
基金
国家自然科学基金(82174399)
江苏省中医药科技发展计划(ZD202230)
苏州市临床重点病种诊疗技术专项(LCZX202320)
苏州市科技发展计划(SKYXD2022053、SKYD2023150)
江苏省医学重点学科和医学重点实验室建设单位(苏卫科教[2022]17号)。
关键词
椎间盘退变
细胞凋亡
Yes相关蛋白
带有PDZ结合基序的转录共激活因子
枳壳甘草汤
intervertebral disc degeneration
apoptosis
yes-associated protein
transcriptional co-activator with PDZ-binding motif
Zhiqiao Gancao Decoction(枳壳甘草汤)
作者简介
通讯作者:沈晓峰,29240818@qq.com。
