The Protective Effects Of Substance P On The Apoptosis Of Corneal Epithelial Cells Induced By High Glucose

PurposeWe cultured mouse corneal epithelial stem and progenitor cells (TKE2) to establishedhigh glucose-induced cell apoptosis model and added substance P (SP) to investigate theeffect of SP on corneal epithelial cells who under the high glucose environment and analysisits possible pathways.Methods1. Established high glucose (HG)-induced apoptosis model of TKE2: TKE2were usedas an experimental model in vitro and cultured in KSFM TKE2basal medium, experiment asfollows:(1)Established concentration gradient of glucose-induced TKE2apoptosis,experimental groups as follows: HG0、50、150、250、350mM，cell apoptosis line were dealtwith Annexin V/PI double staining method and tested by flow cytometry after24h to identifythe best glucose concentration of high sugar-induced TKE2apoptosis.(2)Established treatment time gradient of glucose-induced TKE2apoptosis, Accordingto the results of best glucose concentration in experiment（1）, cells were divided into fourgroups in same glucose concentration:0、12、24、48h，cell apoptosis line were dealt withAnnexin V/PI double staining method and tested by flow cytometry to identify the besttreatment time. 2. Established SP concentration gradient of inhibiting TKE2apoptosis induced by highglucose: according to the high glucose concentration and treatment time identified in methodone, we established treatment concentration gradients of SP, experimental groups asfollows:0、0.1、1、10μM, cell apoptosis line were dealt with Annexin V/PI double stainingmethod in the end and tested by flow cytometry to identify SP has an inhibitory effect on highglucose-induced apoptosis and the best concentration.3. According to the high glucose concentration、treatment time and SP concentrationidentified in method one and two, we investigate possible signaling pathways of SP inhibitioneffect on high glucose-induced apoptosis.(1) SP and receptor NK-1R In the cell culture process by adding NK-1R inhibitorL-733,060(1μM) pretreatment, cell apoptosis line were dealt with Annexin V/PI doublestaining method in the end and tested by flow cytometry, expression levels of Caspase3,8,9were tested by caspase activity detection kit.(2) SP and Akt In the cell culture process, by adding Akt inhibitor (40μM)pretreatment, cell apoptosis line were dealt with Annexin V/PI double staining method in theend and tested by flow cytometry, expression levels of P-Akt and total AKt were detected bywestern blot.(3) SP and oxidative stress Two antioxidants NAC and DPI were uesd as positivecontrol, In the cell culture process, by adding NAC (1mM) and DPI (1μM) two antioxidantspretreatment, cell apoptosis line were dealt with Annexin V/PI double staining method in theend and tested by flow cytometry, ROS staining, calcium fluorescent probe (Fluo-3AM)staining and mitochondrial membrane potential fluorescent probe (JC-1) were performed todetect the change in fluorescence intensity of staining by confocal microscopy andquantitative flow cytometry.Rusults1. High glucose could induce TKE2apoptosis, glucose-induced TKE2apoptosis hadconcentration dependence and time dependence, the effect of glucose-induced TKE2apoptosis was significant in the glucose concentration of250mM and treatment time of24h. 2. SP had an inhibitory effect on high glucose-induced apoptosis that had concentrationdependence, the inhibitory effect was significant when SP concentration was1μM.3. Possible signaling pathways of SP inhibition effect on high glucose-induced apoptosis:(1) SP-NK-1R signaling pathway: the SP group added NK-1R inhibitor L-733,060(1μM), the flow analysis results show HG, HG+SP, HG+SP+NK-1R inhibitor the proportionof apoptotic cells were33.78%,15.68%,34.32%, adding the proportion of NK-1R inhibitorof apoptotic cells was significantly higher than the SP group, the difference was statisticallysignificant (P <0.01). Caspase3,8,9activity assay results shown that compared with highglucose group, expression levels of caspase3,8,9in SP group were significantly lower,difference was statistically significant (P<0.01). SP group added Nk-1Ri, caspase8,9had nodifference (P>0.05) with SP group, but caspase3was significantly increased, and had nodifference (P>0.05) with high glucose group.(2) Akt signaling pathway: the SP group after adding Akt inhibitor (40μM), flowcytometry results are shown HG, HG+SP, HG+SP+ratio of Akt inhibitor of apoptosis cellswere46.70%,15.39%,29.43%, after adding Akt inhibitors, apoptosis was significantlyhigher than the SP group, but still lower than the high-sugar group, each group differenceswere statistically significant (P <0.01). Western Blot results showed compared with thecontrol group and the SP group, expression levels of high glucose group、NK-1R inhibitorgroup and AKt inhibitor group P-Akt were significantly lower, difference was statisticallysignificant (P<0.01), SP group and control There were no differences (P>0.05). The total Aktexpression in each group showed no significant difference (P>0.05).(3) SP and oxidative stress：Antioxidant NAC and DPI were respectively added intohigh glucose groups, compared with high glucose group, in SP group、NAC group and DPIgroup, ROS expression and intracellular calcium concentration decreased significantly(P<0.01), mitochondrial membrane potential increases significantly (P<0.01). High glucosegroup between NK-1Ri group and Akti group showed no significant difference (P>0.05).ConclusionHigh glucose can induce TKE2apoptosis, SP has inhibition effect on highglucose-induced apoptosis. SP inhibit cell apoptosis by connecting with receptor NK-1R, in part through AKt pathway and inhibiting oxidative stress to inhibit apoptosis induced by highglucose.