Effect of Neferine on Apoptosis of Nucleus Pulposus Cells under Abnormal Mechanical Stretch Stress and Its Related Mechanism
10.11842/wst.20250427005
- VernacularTitle:甲基莲心碱对异常机械牵张应力作用下髓核细胞凋亡的影响及相关机制
- Author:
Li ZHANG
1
;
Yi LIU
1
;
Jingmin WANG
1
Author Information
1. 武汉市第四医院 武汉 430033
- Publication Type:Journal Article
- Keywords:
Nucleus pulposus cells;
Mechanical stretch stress;
Neferine;
Apoptosis;
mitochondria;
Reactive oxygen species
- From:
World Science and Technology-Modernization of Traditional Chinese Medicine
2025;27(10):2928-2940
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the effect of neferine(Nef)on the apoptosis of nucleus pulposus cells under abnormal mechanical stretch stress and its related mechanism.Methods Rat nucleus pulposus cells were subjected to cyclic mechanical stretch stress using Flexcell-5000T tension system,and different concentrations of Nef(6.25,12.5,25 μmol/L)and acetylcysteine(NAC,2 mmol/L)were used to intervention cells.CCK-8 was used to detect cell proliferation activity;Hoechst 33258 fluorescence staining was used to observe the morphological changes of the nucleus;Flow cytometry was used to detect the cell apoptosis level;Biochemical kits were used to detect the levels of lactate dehydrogenase(LDH)in the supernatant,malondialdehyde(MDA)and superoxide dismutase(SOD)in the cells;Western blot was used to detect the expression levels of apoptosis-related proteins,including Bax,Bcl-2,apoptotic protease activating factor-1(Apaf-1),Cleaved-caspase 9,Cleaved-caspase 3,as well as extracellular matrix-associated proteins aggrecan cartilage proteoglycan(Aggrecan),collagen type Ⅱ alpha 1 chain(COL2A1),matrix metalloproteinase-3(MMP-3),and antioxidant signaling pathway proteins nuclear factor erythroid 2-related factor 2(Nrf2)and heme oxygenase 1(HO-1).DCFH-DA fluorescent probe was used to detect the level of reactive oxygen species(ROS)in cells.JC-1 staining was used to detect the level of mitochondrial membrane potential in cells.Results The apoptosis rates of nucleus pulposus cells in the control group and the 5%,10%,15%,and 20%tensile strain groups were(6.69±0.37)%,(8.24±0.33)%,(14.45±0.65)%,(30.48±0.78)%,and(43.06±1.32)%,respectively.Compared with the control group,exposure to 5%,10%,15%,and 20%mechanical stretch stress significantly increased the apoptosis rate of nucleus pulposus cells,the level of LDH in the supernatant,and the levels of ROS and MDA within the cells(P<0.05).Additionally,the levels of SOD and mitochondrial membrane potential were significantly reduced(P<0.05).Simultaneously,the protein expression levels of Bax,Apaf-1,Cleaved-caspase 9,and Cleaved-caspase 3 were markedly upregulated(P<0.05),while the expression level of Bcl-2 protein was downregulated(P<0.05).The apoptosis rates of nucleus pulposus cells in the 15%stretch group,15%stretch+6.25 μmol/L Nef,15%stretch+12.5 μmol/L Nef,and 15%stretch+25 μmol/L Nef groups were(29.74±1.03)%,(25.09±0.67)%,(20.57±0.45)%,and(16.06±0.52)%,respectively.Compared with the 15%stretch group,treatment with 6.25,12.5,and 25 μmol/L Nef significantly reduced the apoptosis rate of nucleus pulposus cells,as well as the levels of LDH in the supernatant and intracellular ROS and MDA(P<0.05);Conversely,the SOD activity and mitochondrial membrane potential were significantly enhanced(P<0.05);Additionally,the protein expression levels of Bax,Apaf-1,Cleaved-caspase 9,Cleaved-caspase 3 and MMP-3 were markedly decreased(P<0.05),while the expression level of Bcl-2,Aggrecan,COL2A1,Nrf2 and HO-1 were increased(P<0.05);Notably,the combination of 25 μmol/L Nef and NAC intervention exhibited a more pronounced effect on inhibiting apoptosis in nucleus pulposus cells induced by abnormal mechanical stretch stress(P<0.05).Conclusion Neferine can inhibit the apoptosis of nucleus pulposus cells induced by abnormal mechanical stretch stress,potentially through scavenging ROS and inhibiting mitochondrial apoptosis pathways.
