基于cGAS-STING信号通路探讨脑震宁含药血清对LPS诱导HT22细胞炎症的影响OA

Objective To investigate the regulatory effect of Naozhenning(脑震宁,NZN)-containing serum on lipopolysaccharide(LPS)-induced inflammatory response in HT22 cells,and to clarify its mechanism of action through the cyclic GMP-AMP synthase(cGAS)-stimulator of interferon genes(STING)signaling pathway.Methods First,the optimal working concentrations and durations of LPS and NZN serum on HT22 cells were determined by the CCK-8 assay,followed by grouped treatment.The blank group was cultured routinely;the LPS group was induced for inflammation with LPS-containing medium;the NZN serum group was treated with LPS-containing medium supplemented with NZN serum;the inhibitor group was preconditioned with the STING inhibitor SN-011 prior to cul‑ture in LPS-containing medium;the NZN serum+inhibitor group was first preconditioned with SN-011,then cultured in LPS-containing medium supplemented with NZN serum.Cell proliferation capacity was evaluated by combining the CCK-8 and EdU assays;neuronal inflammatory status was determined using the nitric oxide(NO)detection assay;the positive cell rate of tumor necrosis factor-α(TNF-α)was measured by immunofluorescence staining;inflammatory factors including interleukin-18(IL-18),TNF-α,and inter‑feron-γ(IFN-γ)were quantified via enzyme-linked immunosorbent assay(ELISA);the protein expressions of cGAS,STING,phosphorylated-STING(p-STING),and interferon regulatory factor 3(IRF3)in cells were detected by Western blot(WB);The copy number of mitochondrial DNA(mtDNA)in cells was detected using quantitative real-time polymerase chain reaction(qRT-PCR).Results Compared with the blank group,the relative proliferation rate of HT22 cells in the LPS group was significantly decreased,the NO content,fluorescence-positive rate,and levels of IL-18,TNF-α,and IFN-γ were significantly increased,and the protein expres‑sions of cGAS,STING,p-STING,and IRF3 as well as the copy number of mtDNA were all significantly upregulated(P<0.01).Com‑pared with the LPS group,the relative proliferation rate of HT22 cells in the NZN serum group,inhibitor group,and NZN serum+inhibitor group was significantly increased,the fluorescence-positive rate and levels of IL-18,TNF-α,and IFN-γ were significantly decreased,and the protein expressions of cGAS,STING,and IRF3 as well as the copy number of mtDNA were all significantly down‑regulated(P<0.05 or P<0.01).Conclusion NZN serum can ameliorate the inflammatory status of HT22 cells induced by LPS,and its mechanism of action is presumably mediated by the cGAS-STING signaling pathway.