基于ROS/p38 MAPK信号通路探究除风益损汤干预PM2.5诱导干眼的作用机制OA

Objective:To establish a mouse model of particulate matter 2.5(PM2.5)-induced dry eye and investigate whether Chufeng Yisuntang can ameliorate the PM2.5-induced ocular surface damage by regulating the reactive oxygen species(ROS)/p38 mitogen-activated protein kinase(p38 MAPK)signaling pathway.Methods:Sixty 8-week-old male C57BL/6J mice were used.Ten were randomly selected as the control group.The remaining 50 mice received topical instillation of 1 drop(0.1 mL)of 5 g·L-1 PM2.5 suspension in both eyes,four times daily.Successfully modeled mice were randomized into four groups(n=10):Model,p38 MAPK inhibitor,Chufeng Yisuntang,and combination(Chufeng Yisuntang at 7.3 g·kg-1+p38 MAPK inhibitor SB203580 at 5 mg·kg-1).Chufeng Yisuntang was administered via gavage,and the inhibitor group via intraperitoneal injection.The control and model groups received equal volumes of distilled water by gavage.All treatments lasted for 4 weeks.General conditions were dynamically observed.Tear secretion,tear film break-up time,and corneal fluorescein staining were assessed.After intervention for 4 weeks,hematoxylin and eosin(HE)staining was used to examine the histopathological changes.Enzyme-linked immunosorbent assay(ELISA)was adopted to measure serum levels of ROS,malondialdehyde(MDA),superoxide dismutase(SOD)1,and SOD2.Western blot and Real-time PCR were employed to determine the protein and gene levels,respectively,of p38 MAPK,B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),and cysteinyl aspartate-specific proteinase-3(Caspase-3)in the corneal tissue.Results:Compared with the control group,the model group exhibited reduced tear secretion volume and tear film breakup time,along with increased corneal fluorescein staining scores(P＜0.01).Compared with the model group,the Chufeng Yisuntang group,p38 MAPK inhibitor group,and combination group demonstrated increased tear secretion volume and tear film breakup time,along with decreased corneal fluorescein staining scores(P＜0.01).HE staining revealed that compared with the control group,the model group exhibited marked increases in corneal epithelial cell layers and epithelial thickness,along with reduced meibomian gland acini and intensely stained,densely packed nuclei around the acini.Compared with the model group,the Chufeng Yisuntang group,p38 MAPK inhibitor group,and combination group showed intact corneal structure,improved cell morphology,and reduced damage severity.ELISA revealed elevated ROS and MDA levels(P＜0.01)and decreased SOD1 and SOD2 levels(P＜0.01)in the model group compared with the control group.Compared with the model group,Chufeng Yisuntang,p38 MAPK inhibitor,and the combination lowered ROS and MDA levels(P＜0.01),while raising SOD1 and SOD2 levels(P＜0.05,P＜0.01).Western blot revealed that compared with the control group,the model group exhibited increased protein levels of p38 MAPK,Bax,and Caspase-3(P＜0.01)and reduced protein level of Bcl-2(P＜0.01).Compared with the model group,Chufeng Yisuntang,p38 MAPK inhibitor,and the combination down-regulated the protein levels of p38 MAPK,Bax,and Caspase-3(P＜0.01),while up-regulating the protein level of Bcl-2(P＜0.01).Compared with the Chufeng Yisuntang group,the combination group exhibited decreased protein levels of p38 MAPK,Bax,and Caspase-3(P＜0.01)and increased protein level of Bcl-2(P＜0.01).Real-time PCR revealed that compared with the control group,the model group exhibited upregulated mRNA levels of p38 MAPK,Bax,and Caspase-3(P＜0.01),and downregulated mRNA level of Bcl-2(P＜0.01).Compared with the model group,Chufeng Yisuntang,p38 MAPK inhibitor,and the combination down-regulated the mRNA levels of p38 MAPK,Bax,and Caspase-3(P＜0.01),while up-regulating the mRNA level of Bcl-2(P＜0.05,P＜0.01).Compared with the Chufeng Yisuntang group,the combination group exhibited decreased mRNA levels of p38 MAPK,Bax,and Caspase-3 expression(P＜0.05,P＜0.01)and increased mRNA level of Bcl-2(P＜0.01).Conclusion:Chufeng Yisuntang may partially protect against PM25-induced corneal injury by inhibiting the ROS/p38 MAPK pathway,enhancing antioxidant defense,and reducing epithelial apoptosis.