Material basis and action mechanism of drug-containing serum of Modified Erxian Pill inhibiting macrophage pyroptosis

doi:10.12307/2025.077

Abstract

Abstract: BACKGROUND: Our previous study found that Modified Erxian Pill could alleviate inflammation in collagen-induced arthritis rats, but its mechanism needs to be further verified.
OBJECTIVE: To analyze the components absorbed in the blood of Modified Erxian Pill, and observe the effect of the drug-containing serum of Modified Erxian Pill on pyroptosis of J774A.1 macrophages.
METHODS: (1) Analysis of components absorbed in the blood of Modified Erxian Pill: Ultra-high performance liquid chromatography-high resolution mass spectrometry was used to detect and identify Modified Erxian Pill and its components absorbed in the blood. (2) Effect of the drug-containing serum of Modified Erxian Pill on pyroptosis of J774A.1 macrophages: Molecular docking technology was used to initially verify the sesquiterpenoids and NLRP3 in components absorbed in the blood of Modified Erxian Pill. J774A.1 macrophages were randomly divided into blank control group, lipopolysaccharide+adenosine triphosphate group, and lipopolysaccharide+adenosine triphosphate+Modified Erxian Pill with low (2.5%), medium (5%), and high (10%) dose groups. The release of lactate dehydrogenase in the cell supernatant of each group was detected according to the kit instructions. The levels of interleukin-1β and interleukin-18 in cell supernatant were detected in each group by ELISA. The cell membrane damage was detected by Hoechst/PI staining. The expression levels of NLRP3, Caspase-1, GSDMD, and GSDMD-N protein in the cells of each group were detected by western blot assay.
RESULTS AND CONCLUSION: (1) A total of 32 active components of Modified Erxian Pill were identified, and 21 components entered the blood. The main components into blood included a variety of sesquiterpenoids. (2) Molecular docking results showed that 3-O-Acetyl-13-deoxyphomenone, Incensol oxide, Atractylenolide III, Rupestonic acid, and 3,7-Dihydroxy-9,11-eremophiladien-8-one had good binding activity with NLRP3. (3) Compared with the blank control group, lactate dehydrogenase activity and the expression levels of interleukin-1β and interleukin-18 were significantly increased in cell supernatant of lipopolysaccharide+adenosine triphosphate group (P < 0.001). Hoechst/PI staining showed that the number of PI-positive cells was significantly increased. After the intervention of lipopolysaccharide+adenosine triphosphate+Modified Erxian Pill group, all of them showed different degrees of reduction. (4) Compared with the blank control group, NLRP3, Caspase-1, GSDMD, and GSDMD-N protein expression levels were significantly increased in the lipopolysaccharide+adenosine triphosphate group (P < 0.05). Compared with lipopolysaccharide+adenosine triphosphate group, the protein expressions of NLRP3, Caspase-1, GSDMD, and GSDMD-N were significantly decreased in the lipopolysaccharide+adenosine triphosphate+Modified Erxian Pill group (P < 0.05), and had a certain dose dependence. These findings verify that the drug-containing serum of Modified Erxian Pill may inhibit the pyroptosis of J774A.1 macrophages by regulating the NLRP3/Caspase-1/GSDMD pathway.

Key words: Modified Erxian Pill, drug-containing serum, pyroptosis, J774A.1 macrophage, sesquiterpenoids

CLC Number:

Li Siyuan, Wang Yuru, Xu Ye, Guo Di, Nan Nan, Liu Yang, Zhao Jie, Hao Huiqin. Material basis and action mechanism of drug-containing serum of Modified Erxian Pill inhibiting macrophage pyroptosis[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(19): 4029-4037.

Figures/Tables 5

2.3 MEXP含药血清对J774A.1巨噬细胞活力的影响 CCK-8结果表明，不同体积分数MEXP含药血清干预J774A.1巨噬细胞6，12，24 h时，各含药血清组的细胞活力与完全培养基组相比差异无显著性意义(P > 0.05)，见图4。因此，选择体积分数2.5%，5%，10%分别作为低、中、高含药血清组进行后续实验。 2.4 MEXP含药血清对各组细胞上清中乳酸脱氢酶活性的影响乳酸脱氢酶释放实验显示，与空白对照组相比，脂多糖+三磷酸腺苷组乳酸脱氢酶释放显著升高(P < 0.001)；与脂多糖+三磷酸腺苷组相比，各MEXP含药血清组乳酸脱氢酶释放均显著下降(P < 0.001)，其中5%MEXP含药血清组下降最为明显，见图5。 2.5 MEXP含药血清对各组细胞上清中白细胞介素1β、白细胞介素18水平的影响 ELISA结果显示，与空白对照组相比，脂多糖+三磷酸腺苷组细胞上清中白细胞介素1β和白细胞介素18水平明显升高(P < 0.001)，与脂多糖+三磷酸腺苷组相比，各MEXP含药血清组细胞上清中白细胞介素1β和白细胞介素18水平明显降低(P < 0.01)，见图6。 2.6 MEXP含药血清对细胞Hoechst/PI染色的影响 Hoechst/PI染色结果显示，脂多糖+三磷酸腺苷组较空白对照组PI阳性细胞数量明显增多，而经过MEXP含药血清干预后PI阳性细胞数量明显减少，见图7。 2.7 MEXP含药血清对各组细胞中NLRP3、Caspase-1、GSDMD、GSDMD-N蛋白表达的影响 Western blot结果显示，与空白对照组相比，脂多糖+三磷酸腺苷组NLRP3、GSDMD、Caspase-1、GSDMD-N蛋白表达显著增高(P < 0.05)；与脂多糖+三磷酸腺苷组相比，2.5% MEXP含药血清组GSDMD、Caspase-1、GSDMD-N蛋白表达显著降低(P < 0.05)，5%和10%MEXP含药血清组NLRP3、GSDMD、Caspase-1、GSDMD-N蛋白表达显著降低(P < 0.05)，见图8。"

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