| Objective: As a well-known metabolic disease, diabetes has become thethird leading cause of death worldwide along with changes in the country’sdevelopment and people’s mothod of life. Diabetic retinopathy resulting in anumber of irreversible vision loss every year sustained to rise as the mostserious complications of eyes. Age-related macular degeneration, which is themost common cause of blindness in the elderly population and every threepeople will be affected of AMD aged65or older. Many experimental studiesand clinical trials have shown: oxidative stress and inflammation has becomethe two very important reasons in the development of AMD. The retina itselfis vulnerable to oxidative stress, because the metabolism of oxygenconsumption of the retina is much higher than any other body tissue andsusceptible to oxidation of polyunsaturated fatty acids and the ripple effectwill stimulate cytotoxic. Currently there is a large number of studies haveshown that: Application RPE cells cultured in high glucose medium, analogRPE cells high glucose in vivo and resulting in cellular oxidative stress. Thenintervene with drugs showed that the activity decreased a high glucose andsome changes in the expression of cytokines and inflammatory factors. In thisstudy, different concentrations of cholecystokinin octapeptide and interactwith the cultured rat retinal pigment epithelial cells of high glucose,48hourslater, MTT assay was used to test the cell viability and test the changes inexpression of NRF2, SOD1, γ-GCS, NQO1, TNF-α,IL-1α and IL-6byRT-PCR. According to the results of screening appropriate treatmentconcentration of CCK-8and detected the levels of all the factors after CCK-8worked on RPE cells for24hour,48hour and72hour.Methods:1Selected the therapeutic concentrations of CCK-8: Pick upRPE cells cultured in logarithmic growth phase and divided into eight groups randomly:①Control group(glucose concentration:5.56mmol/L)②High-glucose group (glucose concentration:30mmol/L)③Hypertonic controlgroup(glucose concentration:5.56mmol/L+mannitol:24.44mmol/L)④High-glucose+CCK-8treatment group (glucose concentration:30mmol/L+CCK-8concentration:10-5mmol/L)⑤High-glucose+CCK-8treatmentgroup (glucose concentration:30mmol/L+CCK-8concentration:10-6mmol/L)⑥High-glucose+CCK-8treatment group (glucose concentration:30mmol/L+CCK-8concentration:10-7mmol/L)⑦High-glucose+CCK-8treatmentgroup (glucose concentration:30mmol/L+CCK-8concentration:10-8mmol/L)⑧High-glucose+CCK-8treatment group (glucose concentration:30mmol/L+CCK-8concentration:10-9mmol/L). Applied the same kind of medium firstand replaced with conditioned medium after24hours cultured and thencultured for48hours. Cell morphology was observed under an invertedmicroscope and observed the effects of different concentrations of CCK-8oncell viability by MTT assay. Cell samples were collected at the same time andto detect the expression of NRF2, SOD1, γ-GCS, NQO1, TNF-α, IL-1α andIL-6using RT-PCR.2Factors’ changes in different times of CCK-8concentrations at10-6mmol/L:According to the results of MTT and RT-PCR derived the therapeuticconcentration of CCK-8is10-6mmol/L and divided groups again:①Controlgroup (glucose concentration:5.56mmol/L)②High-glucose (glucoseconcentration:30mmol/L)③Hypertonic control group(glucose concentration:5.56mmol/L+mannitol:24.44mmol/L)④High glucose+CCK-8treatmentgroup (glucose concentration:30mmol/L+CCK-8concentration:10-6mmol/L).The cells in every group were cultured for24hours,48hours and72hoursand detecting expression of the seven factors for RT-PCR.Results:1MTT assay: Compared with normal control group, the viability of cells inhigh-glucose group was significantly lower (P<0.05) and the cell viability inhypertonic control group did not change significantly; cell viability of the treatment groups are lower than control group (P<0.05). There is nosignificant difference among the three groups of CCK-8concentration of10-7mmol/L,10-8mmol/L and10-9mmol/L on cell viability. There is nosignificant difference among the high-glucose group and the above threegroups. While CCK-8at a concentration of10-6mmol/L and10-5mmol/L, cellviability has no significant difference between the two groups, however, thetwo groups’ cell viability are higher than the other three treatment groups andhigh-glucose group (P<0.05).2RT-PCR results of selected therapeutic concentrations of CCK-8: Comparedwith control group, the expression of NRF2, SOD1, γ-GCS, NQO1, TNF-α,IL-1α and IL-6were increased in high glucose group (P<0.05), however,hypertonic control group showed no differences. Compared with the controlgroup, the expression of each treatment groups of NRF2, SOD1, γ-GCS andNQO1were significantly increased (P<0.05) and the expression of TNF-α,IL-1α and IL-6were significantly decreased (P<0.05). The expression ofNRF2, SOD1, γ-GCS and NQO1in CCK-8treatment group at theconcentrations of10-7mmol/L,10-8mmol/L,10-9mmol/L were increasedcompared with the high glucose group (P<0.05), while TNF-α, IL-1α and IL-6were decreased (P<0.05), however there is no significant difference amongthe three groups. There is no significant difference between the two groups ofCCK-8at the concentrations of10-6mmol/L and10-5mmol/L, however,compared with the other three treatment groups and high glucose theexpression of NRF2, SOD1, γ-GCS and NQO1were significantly increased(P<0.05) along with the expression of TNF-α, IL-1α and IL-6significantlydecreased (P<0.05).3Factors’ changes in different times of CCK-8concentrations at10-6mmol/L:Compared with control group, the expression of7factors has no significantdifference in hypertonic control group; the expression of the7factors inhigh-glucose group are significantly increased (P<0.05) compared to thecontrol group and the hypertonic control groups; compared with high-glucosegroup, the expressions of NRF2, SOD1, γ-GCS and NQO1in treatment group were significantly increased (P<0.05) and the level is higher at48hours than24hour and72hours (P<0.05) and there is no significant difference betweenthe24hour and72hours; compared with high-glucose group the expressionof TNF-α, IL-1α and IL-6are significantly decreased (P<0.05) of treatmentgroup and the level is lower at72hour than24hour and48hours (P<0.05)and there is not significant difference between the24hour and48hours.Conclusion:1High-glucose can lead to rat RPE cell viability decreased and damage tocells.2CCK-8at the concentration of10-6mmol/L can inhibited the cells damagescaused by high glucose after a certain time and so the expression of NRF2,SOD1, γ-GCS and NQO1were significantly increased, the expression ofTNF-α, IL-1α and IL-6were significantly decreased, which suggested that theCCK-8may maintain a balance of RPE cells through antioxidant andanti-inflammatory effecting order to protected the cells. All of these establishexperiments foundation for the clinical application of CCK-8in future. |
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