RESUMO
OBJECTIVE: Huangqi Decoction (HQD), a classical traditional Chinese medicine formula, has been used as a valid treatment for alleviating liver fibrosis; however, the underlying molecular mechanism is still unknown. Although our previous studies showed that microRNA-663a (miR-663a) suppresses the proliferation and activation of hepatic stellate cells (HSCs) and the transforming growth factor-ß/small mothers against decapentaplegic (TGF-ß/Smad) pathway, whether long noncoding RNAs (lncRNAs) are involved in HSC activation via the miR-663a/TGF-ß/Smad signaling pathway has not yet reported. The present study aimed to investigate the roles of lncRNA lnc-C18orf26-1 in the activation of HSCs and the mechanism by which HQD inhibits hepatic fibrosis. METHODS: The expression levels of lnc-C18orf26-1, miR-663a and related genes were measured by quantitative reverse transcription-polymerase chain reaction. HSCs were transfected with the miR-663a mimic or inhibitor and lnc-C18orf26-1 small interfering RNAs. The water-soluble tetrazolium salt-1 assay was used to assess the proliferation rate of HSCs. Changes in lncRNA expression were evaluated in miR-663a-overexpressing HSCs by using microarray to identify miR-663a-regulated lncRNAs. RNA hybrid was used to predict the potential miR-663a binding sites on lncRNAs. Luciferase reporter assays further confirmed the interaction between miR-663a and the lncRNA. The expression levels of collagen α-2(I) chain (COL1A2), α-smooth muscle actin (α-SMA) and TGF-ß/Smad signaling pathway-related proteins were determined using Western blotting. RESULTS: Lnc-C18orf26-1 was upregulated in TGF-ß1-activated HSCs and competitively bound to miR-663a. Knockdown of lnc-C18orf26-1 inhibited HSC proliferation and activation, downregulated TGF-ß1-stimulated α-SMA and COL1A2 expression, and inhibited the TGF-ß1/Smad signaling pathway. HQD suppressed the proliferation and activation of HSCs. HQD increased miR-663a expression and decreased lnc-C18orf26-1 expression in HSCs. Further studies showed that HQD inhibited the expression of COL1A2, α-SMA, TGF-ß1, TGF-ß type I receptor (TGF-ßRI) and phosphorylated Smad2 (p-Smad2) in HSCs, and these effects were reversed by miR-663a inhibitor treatment. CONCLUSION: Our study identified lnc-C18orf26-1 and miR-663a as promising therapeutic targets for hepatic fibrosis. HQD inhibits HSC proliferation and activation at least partially by regulating the lnc-C18orf26-1/miR-663a/TGF-ß1/TGF-ßRI/p-Smad2 axis.
Assuntos
Medicamentos de Ervas Chinesas , MicroRNAs , RNA Longo não Codificante , Humanos , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Longo não Codificante/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , MicroRNAs/genética , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Proliferação de Células , Fatores de Crescimento Transformadores/metabolismo , Fatores de Crescimento Transformadores/farmacologiaRESUMO
In this study, the anti-inflammatory mechanism of Ginkgo biloba extract 50 (GBE50) and its mechanism of action on NLRP3 inflammatory corpuscles were observed by primary microglia cells. LPS/ATP was used to stimulate microglia, and the best time for stimulation and optimal concentration of GBE50 were screened. Pro-inflammatory cytokine IL-1ß and TNF-α was determined by ELISA. Western blot was performed to observe the protein expression of NLRP3, ASC, caspase-1 and IL-1ß in cultured primary rat microglia. Effect of GBE50 on NLRP3 inflammatory corpuscle aggregation was detected by CO-IP. GBE50 (10 mg·L⻹) preconditioning could significantly inhibit the expression of LPS (100 µg·L⻹,12 h), ATP (5 mmol·L⻹, 30 min) induced primary microglia corpuscle associated protein, inflammatory corpuscle aggregation, and the release of inflammatory factors IL-6 and TNF-α. These results indicated that GBE50 could inhibit the secretion of inflammatory factors after microglia activation, which is related to down regulating the protein expression and activity of NLRP3 inflammatory corpuscle.