通督活血汤含药血清可抑制椎间盘纤维环细胞的焦亡

Tongdu Huoxue Decoction-medicated serum inhibits pyroptosis in annulus fibrosus cells of the intervertebral disc

背景:腰椎间盘退变是腰椎间盘突出症的主要病理改变,与椎间盘纤维环细胞的程序性死亡密切相关。既往临床研究表明,通督活血汤治疗腰椎间盘突出症疗效显著,其是否通过抑制椎间盘纤维环细胞焦亡起作用值得深入研究。目的:通过观察通督活血汤含药血清对脂多糖/ATP诱导的椎间盘纤维环细胞焦亡的影响,探讨通督活血汤治疗腰椎间盘突出症的可能作用机制。方法:将36只10周龄SD大鼠以通督活血汤灌胃,连续灌胃7 d后腹主动脉取血,制备含药血清。提取4周龄SD大鼠的腰椎间盘,采用机械-酶消化法提取椎间盘纤维环细胞,培养至第2代后采用甲苯胺蓝染色法和Ⅱ型胶原免疫组化染色法进行鉴定。之后将第2代细胞随机分为空白组、模型组、含药血清低、中、高剂量组,除空白组外,其余4组予脂多糖/ATP制造细胞焦亡模型,成模后低、中、高剂量含药血清组分别予5%,10%,20%通督活血汤含药血清干预,空白组不采取任何干预措施。采用MTT法摸索含药血清干预椎间盘纤维环细胞焦亡的最佳干预浓度和最佳干预时间;采用流式细胞术Annexin V FIFT/PI双染测定细胞焦亡率;采用ELISA法检测各组细胞上清液的白细胞介素1β、白细胞介素18的表达量;采用qRT-PCR检测各组半胱氨酸蛋白酶1、Gasdermin D(GSDMD)、NOD样受体蛋白3基因的表达量;采用Western Blot法检测各组半胱氨酸蛋白酶1、GSDMD、NOD样受体蛋白3蛋白的表达量。结果与结论:①MTT结果示:通督活血汤含药血清干预椎间盘纤维环细胞焦亡的最佳干预浓度为10%,最佳干预时间为24 h;②流式细胞术结果示:与空白组相比,其他4组细胞焦亡比率明显增加;与模型组相比,含药血清各剂量组细胞焦亡比率明显下降;与低、高剂量组相比,中剂量组细胞焦亡比率明显下降;③ELISA、qRT-PCR、Western Blot结果示:与空白组相比,其他4组白细胞介素1β、白细胞介素18的表达量、半胱氨酸蛋白酶1、GSDMD、NOD样受体蛋白3的基因及蛋白表达量均显著升高;与模型组相比,含药血清各剂量组均显著降低;各剂量组组间比较,中剂量组表达量显著低于低、高剂量组;以上差异均有显著性意义(P <0.05);④提示通督活血汤含药血清可有效缓解脂多糖/ATP诱导的椎间盘纤维环细胞发生焦亡,减轻炎症反应;中剂量组在延缓细胞焦亡、减轻炎症反应方面疗效最佳。

BACKGROUND:Lumbar disc degeneration is the main pathological change of lumbar disc herniation,which is closely related to the programmed death of intervertebral disc annulus fibrosus cells.Previous clinical studies have shown that Tongdu Huoxue Decoction has a significant effect in the treatment of lumbar disc herniation,and it is worth further studying whether it can play a role by inhibiting the pyrolysis of annulus fibrosus cells.OBJECTIVE:To investigate the inhibitory effect of Tongdu Huoxue Decoction-medicated serum on the pyroptosis of annulus fibrosus cells in the intervertebral disc induced by lipopolysaccharide/adenosine triphosphate(ATP),so as to explore the possible mechanism of Tongdu Huoxue Decoction in the treatment of lumbar disc herniation.METHODS:Thirty-six 10-week-old Spraue-Dawley rats were gavaged with Tongdu Huoxue Decoction,and blood samples were taken from the abdominal aorta after 7-day continuous gavage to prepare drug-containing serum.Four-week-old Sprague-Dawley rats were taken to extract their lumbar intervertebral discs,and then the annulus fibrosus cells were extracted from the intervertebral disc tissues by mechanical-enzymatic digestion.The cells cultured to the second generation were identified by toluidine blue staining and type II collagen immunohistochemical staining.After that,the second-generation cells were randomly divided into blank group,model group,and low-,middle-,and high-dose serum-containing groups.Except for the blank group,the remaining four groups were given lipopolysaccharide/ATP to make the cell pyroptosis model.After modeling,5%,10%,and 20%of Tongdu Huoxue Decoction medicated serum were administrated in the low-,middle-,and high-dose serum-containing groups,respectively,and no intervention measures were taken in the blank group.We then used the MTT method to find the best concentration and time for the intervention of pyroptosis of annulus fibrosus cells with the drug-containing serum.Flow cytometry and Annexin V FIFT/PI double staining were used to measure the rate of cell pyroptosis.ELISA method was used to detect the expression of interleukin-1βand interleukin-18 in cell supernatant.qRT-PCR was used to detect the mRNA expression of Caspase-1,Gasdermin D,and nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3).RESULTS AND CONCLUSION:MTT showed that the best intervention concentration of Tongdu Huoxue Decoction medicated serum for the pyroptosis of annulus fibrosus cells was 10%,and the best intervention time was 24 hours.Flow cytometry results showed that compared with the blank group,the pyroptosis rate in the other four groups was significantly increased(P<0.05).Compared with the model group,the pyroptosis rate in the three drug-containing serum groups was significantly decreased(P<0.05).Compared with the low-and high-dose groups,the pyroptosis rate of the middle-dose group was significantly decreased(P<0.05).Results from ELISA,qRT-PCR,and western blot assay indicated that compared with the blank group,the expression levels of interleukin-1β,interleukin-18,Caspase-1,Gasdermin D,NLRP3 mRNAs and proteins were significantly increased(P<0.05),while compared with the model group,these expression levels were significantly decreased in the three drug-containing serum groups(P<0.05).Moreover,the expression levels of interleukin-1β,interleukin-18,Caspase-1,Gasdermin D,NLRP3 in the middle-dose group were significantly lower than those in the low-and high-dose groups(P<0.05).Therefore,Tongdu Huoxue Decoction-medicated serum can effectively relieve lipopolysaccharide/ATP induced pyroptosis of intervertebral disc annulus fibrosus cells and reduce inflammation.The middle-dose group has the best effect in delaying cell pyroptosis and reducing inflammation.