瑞香素通过含半胱氨酸的天冬氨酸蛋白水解酶通路对胶原诱导性关节炎大鼠成纤维样滑膜细胞增殖与凋亡的影响

目的探讨瑞香素(DAP)通过含半胱氨酸的天冬氨酸蛋白水解酶(caspase)通路对胶原诱导性关节炎(CIA)大鼠成纤维样滑膜细胞(FLS)增殖与凋亡的影响。方法体外培养 CIA-FLS，选取对数生长期细胞以不同浓度 DAP(0、5、10、20、40、80 mg/

Effects of daphnetin on the proliferation and apoptosis of fibroblast-like synoviocytes in rats with collagen-induced arthritis through the cysteine-containing aspartate proteolytic enzyme pathway

Objective To explore the effect of daphnetin (DAP) on the proliferation and apoptosis of fibroblast-like synoviocytes in collagen-induced arthritic rats (CIA-FLS) through the cysteinyl aspartate specific proteinase (caspase) pathway.Methods CIA-FLSs were cultured in vitro, and cells in logarithmic growth phase were selected and treated with different concentrations of DAP (0, 5, 10,20, 40, 80 mg/L) for 48 h. The cell growth state was observed under an inverted microscope, and the cell viability of CIA-FLSs was de. tected by the CCK-8 method. After treatment with DAP (40 mg/L), the relative expression levels of caspase-3, caspase-8 and caspase-9 mRNA in CIA-FLSs were detected by real-time PCR. After CIA-FLS were pretreated with specific irreversible caspase inhibitors (Casp3-In, Casp8-In, Casp9-In, and Pancasp-In) at different concentrations and for different times, the effect of DAP (40 mg/L) on the viability of CIA-FLS cells was detected by the CCK-8 method. CIA-FLSs were pretreated with different caspase inhibitors for 2 h, andthen cotreated with DAP (40 mg/L) for 48 h. The apoptosis morphology of CIA-FLSs was detected by Hoechst 33258 fluorescence stain. ing and Annexin V-FITC/PI double staining.Results The cell viability of CIA-FLSs decreased and the proliferation was significantly inhibited after DAP treatment, and the concentration was in a dose-dependent manner. The relative expression levels of caspase-3, cas. pase-8, and caspase-9 mRNA in CIA-FLSs after DAP treatment were 3.07±0.43,1.67±0.09, and 2.35±0.20, respectively, which weresignificantly higher than those in the control group (P < 0.05). Pretreatment with each caspase inhibitor attenuated the effect of DAP on inhibiting the proliferation of CIA-FLSs to varying degrees, but Casp8-In was not as effective as Casp3-In,Casp9-In, or Pancasp-In. Flu. orescence staining showed that after DAP treatment, the CIA-FLS nuclei had different shapes and sizes, with obvious pyknosis and frag.mentation of the nuclei, and there was more dense granular and massive fluorescence,Casp3-In, Casp9-In and Pancasp-In. Comparedwith the DAP group, the number of normal cell nuclei with uniform blue fluorescence in the pretreatment group was significantly in.creased, and the difference between the Casp8-In pretreatment group and the DAP group was not significant, which was significantly higher than that in the control group (P<0.05). The apoptosis rates of CIA-FLS in the Casp3-In,Casp8-In,Casp9-In, and Pancasp-In pre.treatment groups were (18.09±2.50)%,(29.46±2.16)%,(20.41±3.53)%, and (10.17±1.91)%, respectively, which were significantly lowerthan those in the DAP group (P<0.05),but the CIA-FLS apoptotic rate in the Casp8-In pretreatment group was significantly higher than that in the Casp3-In, Casp9-In, or Pancasp-In pretreatment groups (P<0.05).Conclusion DAP can inhibit the proliferation of CIA-FLSs in a dose-dependent manner and induce CIA-FLS apoptosis through a caspase-dependent pathway, which may be closely related to the mitochondrial apoptosis pathway.