Indoxyl Sulfate Induces Pyroptosis and Inhibits Osteogenic Differentiation in  MC3T3-E1 Cells via Activation of Caspase-8/GSDME Pathway

doi:10.3870/j.issn.1672-0741.25.07.002

Abstract

Abstract: Objective To investigate the mechanism by which indoxyl sulfate(IS)induces pyroptosis in MC3T3-E1 pre-osteoblasts and inhibits osteogenic differentiation through the Caspase-8/GSDME pathway.Methods MC3T3-E1 cells were treated with IS at various concentrations(0,25,50,and 100 μmol/L)for 24,48,and 72 h.Cell proliferative activity was assessed using the CCK-8 assay,and apoptosis was detected by Annexin Ⅴ-FITC/PI flowcytometry.After the optimal concentration and exposure time were selected based on preliminary experiments,the cells were divided into the control group,IS group,and IS combined with the Caspase-8-specific inhibitor Z-IETD-FMK group.Lactate dehydrogenase(LDH)release,Caspase-8/GSDME-related protein expression(Western blot),alkaline phosphatase(ALP)activity,osteogenesis-related protein expression,and calcium nodule formation(ARS staining)were detected in the three groups.Results IS showed dose-dependent cytotoxicity after 48 h of treatment,with the strongest inhibitory effect at 100 μmol/L,significantly reducing cell viability(P<0.01).Western blot analysis showed that IS upregulated the expression of Caspase-8,cleaved Caspase-8,gasdermin E(GSDME),and its cleaved product GSDME-N(all P<0.01),and promoted LDH release(P<0.01).These changes were partially reversed after Z-IETD-FMK intervention(all P<0.05).Regarding osteogenic function,IS significantly inhibited ALP activity on days 3 and 5(both P<0.01),downregulated the mRNA and protein expression levels of osteopontin(OPN),osteocalcin(OCN),and bone sialoprotein(BSP)(P<0.01),and reduced ARS-stained calcium nodule formation(P<0.01).After blockade of the pyroptosis pathway,osteogenesis-related indicators recovered to varying degrees(all P<0.05).Conclusion IS induces pyroptosis in MC3T3-E1 cells by activating the Caspase-8/GSDME pathway,causing cell membrane rupture and functional impairment,thereby inhibiting early and late osteogenic differentiation.This pathway may represent a key molecular mechanism underlying the osteotoxic effects of IS.

Key words: indoxyl sulfate, Caspase-8, GSDME, pyroptosis, osteogenic differentiation, MC3T3-E1 cells

CLC Number:

R681.1

Wang Qian, Cai Jiping, Tian Xiaochen. Indoxyl Sulfate Induces Pyroptosis and Inhibits Osteogenic Differentiation in MC3T3-E1 Cells via Activation of Caspase-8/GSDME Pathway[J]. Acta Medicinae Universitatis Scientiae et Technologiae Huazhong, 2026, 55(4): 490-495.

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Indoxyl Sulfate Induces Pyroptosis and Inhibits Osteogenic Differentiation in MC3T3-E1 Cells via Activation of Caspase-8/GSDME Pathway

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