人乳铁蛋白通过HMGB1/TLR4信号通路减轻内质网应激介导的骨关节炎损伤OA

Objective To investigate the impact of human lactoferrin(HLF)on human articular chondrocytes(HCC)and elucidate the underlying mechanisms by which HLF exerts its effects on rat models for arthritis.Methods The CCK-8 assay was performed to assess the cytotoxicity of various concentrations of HLF,ranging from 1 to 30 μg/mL,and interleukin(IL)-1β,from 5 to 40 ng/mL,on chondro-cytes,thereby establishing appropriate concentrations for subsequent experimental procedures.The cells were divided into the control group(without HLF and IL-1β),model group(with IL-1β alone),and experimental group(with IL-1β and different concentrations of HLF).Western blotting was used to examine protein expression in HCC after HLF treatment.The regulatory mechanism of HLF was validated using the endoplasmic reticulum stress inhibitor 4-PBA and high mobility group box 1(HMGB1)inhibitor EP.Real-time quantitative PCR was performed to quantify the expression levels of related genes.Cell proliferation was visualized using EdU staining.Intracellular reactive oxygen species(ROS)levels were measured using the DCFH-DA fluorescent probe.The rats were divided into the control group(knee joint injection with normal saline),model group(injection of IL-1β and PBS gavage),and experimental group(injection of IL-1β and gavage of HLF).To evaluate articular tissue alterations in rat models for arthritis,histological staining methods,such as toluidine blue,saffron-O-fast green,and hematoxylin-eosin staining,were used.Immunohistochemistry was used to assess protein expression in the rat joint tissues.Results HLF promoted chondrocyte proliferation in a concentration-dependent manner.Specifically,low concentrations of IL-1β(5 and 10 ng/mL)had no significant impact on cell viability,whereas higher concentrations(20,30,and 40 ng/mL)inhibited cell proliferation.Based on these findings,concentrations of 5,10,and 20 μg/mL HLF,along with 10 ng/mL IL-1β,were selected for fur-ther investigation.According to the cell experiment results,compared with the control group,the protein and mRNA expression levels of HMGB1,Toll-like receptor 4(TLR4),prostate-associated gene protein(PAGE),matrix metalloproteinase 3(MMP3),tumor necrosis factor(TNF)-α,and C/EBP homologous protein(CHOP)were significantly upregulated in the model group(all P<0.05).Cell proliferation was inhibited,and ROS levels increased.After treatment with different concentrations of HLF,the expression of the above-mentioned proteins and mRNA was downregulated in a concentration-dependent manner,cell proliferation gradually recovered,and ROS levels significantly decreased(all P<0.05).In addition,treatment with 4-PBA or EP significantly downregulated the expression of related proteins(all P<0.05).The animal experiments showed that HLF improved the histopathological status of the knee joint and reduced the protein expres-sion of HMGB1,TLR4,MMP3,and CHOP in the rats with osteoarthritis.Conclusion HLF significantly promoted the proliferation of HCC,inhibited the expression of related genes and proteins,and enhanced the antioxidant capacity.HLF ameliorated arthritis in the rats.Its mechanism may be related to inhibition of the HMGB1/TLR4 signaling pathway and alleviation of endoplasmic reticulum stress and inflammatory responses.