| Objective: To observe the role of p38 MAPK signaling pathway mediated cell senescence in the pathogenesis of conjunctivochalasis(CCH),and to study the protective effect of Qi Jing Mingmu Decoction on p38 ediated CCH cell senescence,so as to provide a new idea for the exploration and treatment of the pathogenesis of CCH.Methods: 1.CCH conjunctival fibroblasts were cultured and transfected with lentiviral vector p38 by RNA interference.The expression of p38? was detected by q-PCR and Western blot to screen effective interference fragments.2.CCH and normal human conjunctival fibroblasts were cultured and treated with RNA interference and p38 inhibitor SB203580 respectively.They were divided into five groups(Control,CCH,CCH+si NC,CCH+siP38,CCH+SB203580).CCK-8 was used to detect cell proliferation,?-gal staining was used to detect cell senescence,flow cytometry was used to detect reactive oxygen species(ROS),spectrophotometry was used to detect superoxide dismutase(SOD)activity,q-PCR and Western blot were used to detect the expression of p38?,p53,p21,SMP30.3.CCH and normal human conjunctival fibroblasts were cultured and treated with Qi Jing Mingmu Decoction granule and p38 inhibitor SB203580 respectively.They were divided into four groups(Control,CCH,CCH+QJMM,CCH+SB203580).Cell proliferation was detected by CCK-8,ROS was detected by flow cytometry,SOD activity was detected by spectrophotometry,and expression of p38?,p53,p21 and SMP30 was detected by q-PCR and Western blot.The double luciferase reporter gene was used to detect the effect of Qi Jing Mingmu Decoction on p38 promoter.4.Forty patients(80 eyes)with CCH of grade II-III liver-kidney Yin deficiency were randomly divided into Qi Jing Mingmu Decoction group and artificial tear group.They were treated with Qi Jing Mingmu Decoction combined with artificial tear and simple artificial tear respectively.The international ocular surface disease index(OSDI),tear break-up time(BUT)and Schirmer ? test(SIT)were observed and clinical therapeutic effects were compared after 3 months treatment.For the patients with CCH grade III or above,follow-up for 3 months or more,the loose conjunctival tissues were removed and frozen sections were observed by ?-gal staining.Results: 1.The expression of p38? mRNA and protein in CCH conjunctival fibroblasts was significantly down-regulated after RNA interference,and siRNA lentivirus vector containing target p38 gene was successfully constructed.2.?-gal staining showed that the cell senescence of conjunctival fibroblasts in CCH group was higher than that in Control group(P=0.001).CCK-8 results showed that OD values of CCH and CCH+si NC group after 48 hours were lower than those of Control group,while OD values of CCH+siP38 and CCH+SB203580 groups were higher than those of CCH and CCH+si NC groups(all P<0.05).ROS content in CCH and CCH+si NC groups was higher than that in Control group,while CCH+siP38 and CCH+SB203580 groups were lower than that in CCH and CCH+si NC groups,but higher than that in Control group(all P<0.05).SOD activity in CCH and CCH+si NC groups was lower than that in Control group,and CCH+siP38 and CCH+SB203580 groups were higher than that in CCH and CCH+si NC groups(all P<0.01).The expression of p38?,p53,p21 mRNA in CCH and CCH+si NC groups was higher than that in Control group,while the expression of SMP30 mRNA was down-regulated(all P<0.01).The expression of p38?,p53 and p21 mRNA in CCH+siP38 and CCH+SB203580 groups was lower than that in CCH and CCH+si NC groups,while the expression of SMP30 mRNA was up-regulated(all P<0.05).The expression of p38?,p53,p21 protein in CCH and CCH+si NC groups was higher than that in Control group,while the expression of SMP30 protein was down-regulated(all P<0.01).The expression of p38?,p53,p21 protein was lower in CCH+siP38 and CCH+SB203580 groups than that in CCH and CCH+si NC groups,while the expression of SMP30 protein was up-regulated(all P<0.01).3.CCK-8 results showed that OD value of CCH group after 24 hours was lower than that of Control group(P<0.05).The OD value of CCH group on 48 hours was lower than that of Control group,and the OD values of CCH+QJMM and CCH+SB203580 groups were higher than those of CCH group(all P<0.01).ROS content in CCH group was higher than that in Control group,while CCH+QJMM and CCH+SB203580 groups were lower than that in CCH group(all P<0.05).SOD activity in CCH group was lower than that in Control group,and CCH+QJMM and CCH+SB203580 groups were higher than that in CCH group(all P<0.01).The expression of p38?,p53,p21 mRNA in CCH group was higher than that in Control group,while the expression of SMP30 mRNA was down-regulated.The expression of p38?,p53 and p21 mRNA in CCH+QJMM and CCH+SB203580 groups was lower than that in CCH group,while the expression of SMP30 mRNA was up-regulated(all P<0.05).p38?,p-p38 a,p53 and p21 proteins in CCH group were higher than those in Control group,while CCH+QJMM and CCH+SB203580 groups were lower than those in CCH group(all P<0.01).SMP30 protein in CCH group was lower than that in Control group,while SMP30 protein in CCH+QJMM and CCH+SB203580 group were higher than that in CCH group(all P<0.01).Double luciferase reporter gene assay showed that the luciferase activity of CCH+QJMM group was lower than that of CCH group containing p38 promoter(P<0.001).4.Clinical study: The OSDI score,BUT and SIT of Qi Jing Mingmu Decoction group were significantly better than those of artificial tear group.After 3 months of treatment,7 cases(7 eyes)in artificial tear group and 4 cases(4 eyes)in Qi Jing Mingmu Decoction group were treated with surgery.The positive staining rate of senescence cells in Qi Jing Mingmu Decoction group was lower than that in artificial tear group(P=0.013).Conclusions: 1.The successful construction of siRNA lentiviral vector containing target p38 gene provides experimental basis for subsequent research.2.Oxidative stress damage occurs in CCH,which activates the p38 MAPK signaling pathway and its downstream senescence-related factors,and decreases cell proliferation,leading to cell senescence.Silencing or inhibiting the p38 MAPK signaling pathway can down-regulate the expression of p38 MAPK and its downstream senescence-related factors,alleviate oxidative stress injury,promote cell proliferation,and thus delay cell senescence.3.Qi Jing Mingmu Decoction can down-regulate the expression of p38 MAPK and its downstream senescence-related factors,down-regulate the activity of p38 MAPK promoter,alleviate oxidative stress injury,enhance the antioxidant capacity of conjunctival fibroblasts,promote the proliferation of conjunctival fibroblasts,and thus treat CCH.4.Combined with Qi Jing Mingmu Decoction to treat CCH is more effective than simple artificial tear in relieving eye symptoms,improving tear film and promoting tear secretion,and reducing cell senescence in conjunctival tissue,which can be used as a safe and effective treatment besides surgical treatment. |
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