Mechanism of Bazhen Lihe Kangxian Formula (八珍荔核抗纤方) in Regulating HSC-T6 Cells via the NLRP3/GSDMD Signaling Pathway-Mediated Pyroptosis

Author：GONG Junwen1,2, DENG Chang1,2, LIAO Dandan1,2, QIN Kanhui1,2, LUO Weisheng1

Unit：1.Guangxi University of Chinese Medicine, Nanning Guangxi 530200, China;; 2.Guangxi Key Laboratory of Molecular in Traditional Chinese Medicine Prevention and Treatment, Nanning Guangxi 530023, China

Quote：引用：龚俊文，邓嫦，廖丹丹，覃阚慧，罗伟生.八珍荔核抗纤方调控NLRP3/GSDMD信号通路介导细胞焦亡对HSC-T6细胞的作用机制[J].中医药导报,2026,32(2):14-18,23.

DOI：10.13862/j.cn43-1446/r.2026.02.003

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Abstract：

Objective: To investigate whether Bazhen Lihe Kangxian formula (BZLHKXF) affects the progression of hepatic fibrosis by regulating the (nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3,NLRP3)/GSDMD signaling pathway and mediating pyroptosis. Methods: Rats were randomly divided into a blank group and low-dose, medium-dose, and high-dose BZLHKXF groups. The blank group received normal saline by gavage, while the low-dose, medium-dose, and high-dose BZLHKXF groups were administered BZLHKXF granules suspended in normal saline at doses of 0.796, 1.593, and 3.186 g/mL, respectively, to prepare blank serum and drug-containing sera of different doses. The effects of BZLHKXF-containing serum on rh TGF-β1-activated HSC-T6 cells were assessed using the CCK-8 assay, and the cell inhibition rate was calculated. Cellular morphology was observed by transmission electron microscopy (TEM). The levels of IL-1β and IL-18 in the cell supernatant were measured by enzyme linked immunosorbent assay (ELISA). The protein and mRNA expression of NLRP3, GSDMD, and CASPASE-1 in HSC-T6 cells was detected by Western blotting and RT-qPCR, respectively. Results: CCK-8 results showed that low-dose, medium-dose, and high-dose BZLHKXF inhibited the proliferation of rh TGF-β1-treated HSC-T6 cells (P<0.05). The inhibitory effect was dose-dependent and more pronounced at 48 h than at 24 h (P<0.05). TEM revealed that compared with the blank group, cells in the model group and low-dose, medium-dose, and high-dose BZLHKXF groups exhibited varying degrees of swelling, plasma membrane rupture, and perforation. Compared with the model group, cell swelling and membrane perforation were alleviated in low-dose, medium-dose, and high-dose BZLHKXF groups. ELISA indicated that IL-1β and IL-18 levels increased in the model group compared with the blank group (P<0.05). Compared with the model group, IL-1β and IL-18 levels decreased in the low-dose, medium-dose, and high-dose BZLHKXF groups (P<0.05). The effect was dose-dependent. Western blotting results showed that, compared with the blank group, the expression levels of IL-1β and IL-18 in the model group were significantly increased (P<0.05). In comparison with the model group, the expression levels of IL-1β and IL-18 were significantly reduced in the low-dose, medium-dose, and high-dose BZLHKXF group (P<0.05), exhibiting a concentration-dependent decrease. RT-qPCR results showed that, compared with the blank group, the expression levels of NLRP3 mRNA, GSDMD mRNA, and CASPASE-1 mRNA were significantly elevated in the cell model group (P<0.05). Following treatment with BZLHKXF, the  expression levels of NLRP3 mRNA, GSDMD mRNA, and CASPASE-1 mRNA were significantly decreased in the low-dose, medium-dose, and high-dose BZLHKXF groups compared with the cell model group, with the most pronounced reduction observed in the high-dose BZLHKXF group, exhibiting a dose-dependent decrease (P<0.05). Conclusion: Bazhen Lihe Kangxian formula may suppress pyroptosis, inhibit the proliferation and activation of HSC-T6 cells, and reduce inflammatory responses by down-regulating the expression of NLRP3, GSDMD, and CASPASE-1 proteins and mRNAs via the NLRP3/GSDMD signaling pathway, thereby exerting anti-hepatic fibrosis effects.

Key words：hepatic fibrosis; Bazhen Lihe Kangxian Formula; NLRP3/GSDMD signaling pathway; pyroptosis; proliferation

摘要：

目的：探讨八珍荔核抗纤方是否可通过调控核苷酸结合结构域富含亮氨酸重复序列和含热蛋白结构域受体3（NLRP3）/焦孔素D（GSDMD）信号通路介导细胞焦亡影响肝纤维化的进程。方法：将大鼠随机分为空白组及八珍荔核抗纤方低、中、高剂量组，空白组以生理盐水灌胃，八珍荔核抗纤方低、中、高剂量组分别按照0.796 g/mL、1.593 g/mL、3.186 g/mL以生理盐水配八珍荔核抗纤方颗粒进行灌胃，以制备空白血清及八珍荔核抗纤方不同剂量含药血清。通过CCK8法测定不同剂量的八珍荔核抗纤方含药血清对rh TGF-β1激活的HSC-T6细胞的影响。透射电镜观察各组细胞形态。ELISA检测细胞上清液中IL-1β和IL-18含量。采用蛋白印迹（Western blotting）法和RT-qPCR检测HSC-T6细胞中NLRP3、GSDMD和CASPASE-1蛋白的表达及mRNA的表达。结果：CCK8结果显示，八珍荔核抗纤方低、中、高剂量组均能抑制经rhTGF-β1处理的HSC-T6细胞的增殖（P＜0.05），随着八珍荔核抗纤方剂量的增加，抑制增殖作用越明显，且48 h较24 h效果更佳（P＜0.05）。透射电镜结果发现，与空白组比较，模型组及八珍荔核抗纤方低、中、高剂量组细胞均有不同程度的细胞肿胀膨大，细胞质膜破裂，细胞膜穿孔。与模型组比较，八珍荔核抗纤方低、中、高剂量组细胞肿胀程度改善，细胞膜穿孔减少。ELISA结果显示，与空白组比较，模型组中的IL-1β、IL-18含量增加（P＜0.05）；与模型组比较，八珍荔核抗纤方低、中、高剂量组中的IL-1β、IL-18含量均减少（P＜0.05），呈浓度依赖性降低。Western blotting结果表明，与空白组比较，模型组中的IL-1β、IL-18含量增加（P＜0.05）；与模型组比较，八珍荔核抗纤方低、中、高剂量组中的IL-1β、IL-18含量均减少（P＜0.05），呈浓度依赖性降低。RT-qPCR结果显示，与空白组比较，细胞模型组的NLRP3 mRNA、GSDMD mRNA、CASPASE-1 mRNA表达水平明显升高（P＜0.05）；八珍荔核抗纤方处理后，与细胞模型组比较，八珍荔核抗纤方低、中、高剂量组NLRP3 mRNA、GSDMD mRNA、CASPASE-1 mRNA表达水平显著下调，其中八珍荔核抗纤方高剂量组下调最明显，且呈剂量依赖性降低（P＜0.05）。结论：八珍荔核抗纤方可能通过NLRP3/GSDMD信号通路，下调NLRP3、GSDMD、CASPASE-1的蛋白表达及mRNA表达，减少细胞焦亡，抑制HSC-T6细胞增殖活化，降低炎症反应。

关键词：肝纤维化；八珍荔核抗纤方；NLRP3/GSDMD信号通路；细胞焦亡；增殖

Release time：2026-03-05

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