Study on Yijing Decoction Regulating IL-6/STAT3/ERK Pathway to Inhibit Damage to an iBRB Model under High-glucose Conditions
OBJECTIVE To investigate the effect and mechanism of Yijing Decoction on an in vitro blood-retinal barrier(iBRB)model under high-glucose conditions.METHODS An in vitro iBRB model was established through co-culture of rat retinal microvascular pericytes and endothelial cells(rRMPs-rRMECs).The study included three groups,control group(CG),model group(MG),and Yijing Decoction group(YG).The medium for each group contained 5.5 mmol/L glucose,33.0 mmol/L glucose,and 4 mg/mL Yijing Decoction+33.0 mmol/L glucose,respectively,and the cells were cultured for 48 hours.Cell viability was assessed by the Cell Counting Kit-8(CCK-8)assay.Retinal microvascular cell apoptosis was observed using terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL).The mRNA expression levels of interleukin-6(IL-6),signal transducer and activator of transcription 3(STAT3),and extracellular signal-regulated kinase(ERK)were measured by real-time quantitative PCR(RT-qPCR).Protein expression levels of IL-6,phosphorylated STAT3(pSTAT3),phosphorylated ERK(pERK),and tight junction proteins were determined by Western Blot.The morphology of the retinal three-dimensional microvascular model was observed by laser confocal microscopy.RESULTS(1)Cell viability:The cell viability in the MG was significantly lower than that in the CG(t=3.016,P=0.009),whereas it was significantly higher in the YG compared to the MG(t=11.810,P=0.000).(2)Cell apoptosis:The apoptosis rate in the MG was significantly higher than that in the CG(t=54.609,P=0.000),while it was significantly lower in the YG compared to the MG(t=31.455,P=0.000).(3)mRNA expression:The mRNA expression levels of IL-6,STAT3,and ERK in rRMECs-rRMPs were significantly higher in the MG than in the CG(tIL-6=13.097,tSTAT3=29.032,tERK=11.005,all P=0.000).The expression levels of IL-6,STAT3,and ERK mRNA were significantly lower in the YG compared to the MG(tIL-6=4.594,P=0.004;tSTAT3=20.695,P=0.000;tERK=4.066,P=0.020).(4)Protein Expression:The protein expression levels of IL-6,p-STAT3,and p-ERK in rRMECs-rRMPs were significantly higher in the MG than in the CG(tIL-6=6.009,P=0.001;tpERK=7.816,tpSTAT3=11.741,both P=0.000),whereas the expression levels of tight junction proteins ZO-1,Occludin,and Claudin-5 were significantly lower in the MG than in the CG(tZO-1=6.818,tOccludin=6.710,both P=0.001;tClaudin-5=11.766,P=0.000).In the YG,IL-6,p-STAT3,and p-ERK protein expression levels were significantly lower than in the MG(tIL-6=2.973,P=0.025;tpSTAT3=6.685,P=0.001;tpERK=3.859,P=0.008),while the expression levels of ZO-1,Occludin,and Claudin-5 were significantly higher than in the MG(tZO-1=2.640,P=0.039;tOccludin=3.397,P=0.016;tClaudin-5=6.241,P=0.001).(5)Microvascular model morphology:In the CG,rRMECs were tightly enveloped by rRMPs,maintaining an intact vascular-like structure.In the YG,the microvascular model began to disintegrate,accompanied by a decrease in rRMP coverage.The MG retained only a small,incomplete reticular structure,with rRMPs nearly absent.CONCLUSIONS Yijing Decoction can enhance the viability of rRMECs-rRMPs and the expression of tight junction proteins under high-glucose conditions,reduce apoptosis rate,and prevent the morphological destruction of an in vitro three-dimensional microvascular model induced by high glucose.Its mechanism may be related to the regulation of the IL-6/STAT3/ERK pathway.
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