[Mechanism of Tianwang Buxin Dan medicated serum in alleviating adenosine-induced neuroinflammation and neuronal apoptosis via modulation of Trpv1/AMPK pathway].

Abstract

This study aimed to investigate whether the medicated serum of Tianwang Buxin Dan(TWBXD) alleviates adenosine(Ado)-induced neuroinflammation and apoptosis in a co-culture system of neuronal(SK-N-SH) and astrocytic(SVG p12) cells by modulating the transient receptor potential vanilloid subfamily member 1(Trpv1)/adenosine monophosphate-activated protein kinase(AMPK) signaling pathway. The optimal Ado concentration for model establishment and the most effective TWBXD serum concentration were determined using a CCK-8 assay. Quantitative real-time polymerase chain reaction(qRT-PCR) was used to screen and validate the most efficient Trpv1 interference and overexpression plasmids. Experimental groups included control, Ado, Ado + ShTrpv1, Ado + OE-Trpv1, OE-Trpv1, Ado + TWBXD, and OE-Trpv1 + TWBXD groups. Apoptosis was assessed by flow cytometry. Levels of interleukin-1β(IL-1β), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) were measured using enzyme-linked immunosorbent assay(ELISA). Immunofluorescence staining was performed to detect the expression of Trpv1 and AMPK, while Western blot analysis was used to determine the protein levels of Trpv1, AMPK-α, and nuclear factor kappa-B p65(NF-κB p65). The results showed that an inflammatory co-culture model was successfully established after exposure to 3.2 mmol·L~(-1) Ado for 48 h, and 10% TWBXD medicated serum for 48 h yielded the highest neuronal proliferation rate. Compared with the control group, the Ado group exhibited a significantly increased apoptosis rate, elevated IL-1β, IL-6, and TNF-α levels, and markedly enhanced relative fluorescence intensities of Trpv1 and AMPK and protein expression of Trpv1, p-NF-κB p65, and p-AMPKα. Compared with the Ado group, the Ado + ShTrpv1 group showed reduced apoptosis rate, significantly lowered IL-1β, IL-6, and TNF-α levels, decreased relative fluorescence intensities of Trpv1 and AMPK, and markedly downregulated protein expression of Trpv1, p-NF-κB p65, and p-AMPKα. The Ado + OE-Trpv1 group exhibited an enhanced apoptosis rate, significantly increased IL-1β, IL-6, and TNF-α levels, elevated relative fluorescence intensities of Trpv1 and AMPK, and upregulated protein expression of Trpv1, p-NF-κB p65, and p-AMPKα. The OE-Trpv1 + TWBXD group presented a decreased apoptosis rate, lowered IL-1β, IL-6, and TNF-α levels, reduced relative fluorescence intensities of Trpv1 and AMPK, and downregulated protein expression of Trpv1, p-NF-κB p65, and p-AMPKα. In conclusion, TWBXD medicated serum effectively alleviates Ado-induced neuroinflammation and neuronal apoptosis by inhibiting the Trpv1/AMPK signaling pathway, thereby ameliorating neuroinflammation in insomnia.