[Mechanism of Angelicae Pubescentis Radix-Taxilli Herba components in preventing nucleus pulposus cell apoptosis via regulating PINK1/Parkin and Nrf2 pathways based on "Qu-Zhi-Bu-Yi" theory].

内容摘要

Based on the theory of ″Qu-Zhi-Bu-Yi″, this study aimed to investigate the optimal compatibility ratio of Angelicae Pubescentis Radix and Taxilli Herba for preventing and treating pressure-induced human nucleus pulposus(NP) cell apoptosis, and to elucidate the mechanisms. A human NP cell apoptosis model was established using abnormal mechanical pressure at 1.0 MPa. The concentration ratios of quercetin, osthole, and columbianadin were screened by the orthogonal experimental design. Cell proliferation and apoptosis were determined using the cell counting kit-8(CCK-8) assay and Annexin V-APC/PI double staining, respectively. Western blot was performed to detect apoptosis-related proteins, as well as key protein expressions in the phosphatase and tensin homolog(PTEN)-induced putative kinase 1(PINK1)/Parkin-mediated mitophagy pathway, and the nuclear factor erythroid 2-related factor 2(Nrf2)-mediated antioxidant pathway. Mitochondrial ultrastructure was observed by transmission electron microscopy(TEM). Mitochondrial membrane potential was measured using the JC-1 staining. Oxidative stress markers were evaluated by the enzyme-linked immunosorbent assay(ELISA). Energy metabolism was quantified using colorimetry. Reactive oxygen species(ROS) levels were detected using the 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA) fluorescent probe. The Nrf2 overexpression and knockdown model construction efficacy in NP cells was validated by quantitative real-time polymerase chain reaction(qRT-PCR). Additionally, rescue experiments were designed to validate the mechanism of the above pathways. Based on the results, the optimal combination of quercetin at 50 μmol·L~(-1), osthole at 6.25 μmol·L~(-1), and columbianadin at 12.5 μmol·L~(-1) significantly promoted NP cell proliferation and inhibited apoptosis. The components enhanced mitophagy by activating the PINK1/Parkin pathway and alleviated oxidative stress by activating the Nrf2 pathway. Rescue experiments showed that the anti-apoptotic effects of the traditional Chinese medicine(TCM) components could be reversed by mitophagy inhibition and Nrf2 knockdown. In conclusion, the Angelicae Pubescentis Radix-Taxilli Herba components inhibit pressure-induced NP cell apoptosis by synergistically activating PINK1/Parkin-mediated mitophagy and Nrf2-mediated antioxidant response. These findings provide experimental evidence for intervertebral disc degeneration treatment.