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Effect of Zhuangyao Tongluo Formula(壮腰通络方) Containing Serum on TNF-α-mediated Vicious Circle of Pyroptosis of Nucleus Pulposus Cells / 中医杂志
Article in Zh | WPRIM | ID: wpr-997289
Responsible library: WPRO
ABSTRACT
ObjectiveTo explore the mechanism of Zhuangyao Tongluo Formula(壮腰通络方,ZTF) in delaying intervertebral disc degeneration. MethodsM1 macrophages were induced from THP-1 cells using LPS, IFN-γ and PMA. The induced M1 macrophages were then co-cultured with nucleus pulposus cells in a transwell system. Fetal bovine serum was used as the control serum, and the effects of different concentrations (5%, 10%, 15%, 20%) of serum from rats treated with ZTF on the activity of M1 macrophages and nucleus pulposus cells were analyzed using MTT assay. Experiment 1 was established, including the nucleus pulposus cell control group, M1 macrophage control group, nucleus pulposus cell + ZTF group, nucleus pulposus cell + TNF control group, nucleus pulposus cell + TNF + ZTF group, co-culture group, and co-culture + ZTF group. The levels of IL-1β, and IL-18 in the culture supernatant were detected using ELISA. The mRNA expression of IL-1β and IL-18 in nucleus pulposus cells was detected using qPCR. Additionally, the expression of GSDMD protein in nucleus pulposus cells was detected using cell immunofluorescence. In experiment 2, co-culture groups were constructed using TNF-α overexpression (OE) or empty vector (EV) plasmids, including co-culture group, TNF-EV + co-culture group, TNF-EV co-culture group + ZTF, co-culture + ZTF group, TNF-OE co-culture group + ZTF, and TNF-OE + co-culture group. The mRNA and protein expression of TNF-α in M1 cells in each group were detected using qPCR and WB. ResultsThe ZTF with 10% serum was selected for subsequent experiments. The results of experiment 1 showed that compared to the control group of nucleus pulposus cells, there was no statistically significant difference in the levels of IL-1β, IL-18, mRNA, and GSDMD expression in the nucleus pulposus cells + ZTF group (P>0.05). However, the TNF-α + co-culture group showed a significant increase in IL-1β, IL-18 levels, mRNA, and GSDMD expression (P<0.01). When compared to the co-culture group, the ZTF+ co-culture group showed a significant decrease in IL-1β, IL-18 levels, mRNA, and GSDMD expression (P<0.01). The results of experiment 2 showed that there was no statistically significant difference in TNF-α mRNA and protein expression between the empty vector plasmids + co-culture group and the co-culture group (P>0.05). Compared to the empty vector + co-culture group, the expression of TNF-α mRNA and protein was significantly reduced in the empty vector co-culture + ZTF group (P<0.01). Compared to the co-culture group and the empty vector + co-culture group, the expression of TNF-α mRNA and protein was significantly reduced in the co-culture + ZTF group (P<0.01). Compared to the co-culture + ZTF group, the expression of TNF-α mRNA and protein significantly increased in the overexpression vector co-culture + ZTF group (P<0.01). Compared to the overexpression vector co-culture + ZTF group, the expression of TNF-α mRNA and protein significantly increased in the overexpression vector co-culture group (P<0.01). ConclusionZTF serum can inhibit the TNF-α-induced apoptosis of nucleus pulposus cells and delay lumbar disc degeneration by reducing the expression of TNF-α in M1 macrophages.
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Full text: 1 Index: WPRIM Language: Zh Journal: Journal of Traditional Chinese Medicine Year: 2023 Type: Article
Full text: 1 Index: WPRIM Language: Zh Journal: Journal of Traditional Chinese Medicine Year: 2023 Type: Article