Effect Of Gigantol On The TNF-?/NF-?B Pathway In Diabetic Retinopathy

Objective: By observing the effects of Gigantol on the diabetic model of C57BL/6J mice induced by streptozotocin and the RF/6A cells cultured in high glucose or TNF-? environment in vitro,to investigate the effects of Gigantol on the TNF-?/NF-?B pathway in diabetic retinopathy.Methods: 1 Experiment in vivo: The diabetic model of C57BL/6J male mice induced by STZ was divided into four groups by random number table.Control group(n=10),diabetic group(n=10),diabetes + 3 mg/kg Gigantol group(n=10),diabetes + 30 mg/kg Gigantol group(n=10).After the models were completed and continued feeding for 8 weeks,then give gigantol was injected with intervention for 1 months.HE staining was used to observe the structure of retinal tissue.The expression of TNF-? and IL-6 in serum of mice was detected by ELISA kit.The effect of Gigantol on the related proteins in TNF-?/NF-?B signaling pathway in mice's retina was detected by Western boltting.Real-time PCR was used to observe the m RNA levels of pro-inflammatory factors TNF-?,IL-6 and IL1-? in mice's retina.The effect of Gigantol on NF-?B protein in mice's retina were observed by immunohistochemistry.2 Experiment in vitro:(1)Using CCK-8 kit to detect the proliferation/toxicity of Gigantol on RF/6A cells,and set the concentration of Gigantol at 0 ?M,0.625 ?M,1.5 ?M,3 ?M,6.25 ?M,12.5 ?M,25 ?M,50 ?M,and 100 ?M.This selection is suitable for the range of drug concentrations.(2)The dual luciferase reporter gene system was used to analyze whether Gigantol can regulate the transcriptional activity of NF-?B.(3)RF/6A cells were cultured in high glucose(30mmol/L)or TNF-?(5ng/ml)environment in vitro.The effect of Gigantol on the expression of proinflammatory cytokines TNF-?,IL-6 and IL1-? m RNA in RF/6A cells in high glucose environment were observed by Real-time quantitative PCR.Western boltting was used to detect the effects of Gigantol on the changes of related proteins in TNF-?/NF-?B signaling pathway in RF/6A cells exposed to high sugar or TNF-?.Results: 1 Experiment in vivo:(1)HE staining of retina in the DM group showed that the retinal stratification was not clear,the retinal inner membrane was swollen,the thickness of the inner and outer nuclear layers was reduced,the cells were arranged disorderly,and the ganglion cells showed vacuolization changes.Different doses of the Gigantol-treated group compared with the DM group,the stratification of the retinal tissue was clearer,the number of inner and outer nuclear layers increased,the cell morphology was slightly regular,the arrangement was mildly disordered,and the swelling of the ganglion cell layer was alleviated.(2)Compared with the control group,the serum levels of TNF-? and IL-6 in the DM group were significantly higher.;Compared with the DM group,the different doses of the group of the Gigantol group could reduce the serum TNF-? and IL-6 expression level.(3)Western boltting's results showed that the expression of TNF-?,NF-?B p65 and its phosphorylated p65 protein in retinal tissue was significantly increased compared with control group;Compared with DM group,different doses of Gigantol can inhibit the expression of TNF-?,NF-?B p65 and its phosphorylated p65 protein in retinal tissue.(4)The results of RT-PCR showed that the m RNA expression levels of TNF-?,IL-6 and IL1-? in retinal tissue were significantly higher than those in Control group;Compared with the DM group,different doses of the group of s Gigantol can decrease the m RNA expression levels of TNF-?,IL-6 and IL1-? in the retina tissue.(5)The results of immunohistochemistry showed that NF-?B was positively expressed in mice's retina,but the expression of NF-?B was significantly increased in DM group compared with Control group;Compared with DM group,the expression of NF-?B was significantly decreased in different doses of Gigantol group,and there was a dosedependent relationship.2 Experiment in vitro:(1)The results of CCK-8 showed that the activity of RF/6A cells was not inhibited when the concentration of Gigantol was less than 50?M.The activity of RF/6A cells was the highest when the concentration of Gigantol was 25?M.The concentration of 5?M,10?M and 25?M Gigantol was used for subsequent experiments.(2)The results of the dual luciferase reporter gene system showed that Gigantol inhibited the transcriptional activity of NF-?B.(3)The results of RT-PCR showed that Gigantol can down-regulate the expression of proinflammatory factors TNF-?,IL-6 and IL-1? m RNA in RF/6A cells under high glucose environment.(4)The results of Western blotting showed that in the high glucose or TNF-? environment,Gigantol can up-regulate the expression of IKb? protein in RF/6A cells and downregulate the expression of NF-?B p65 and p-p65 protein.Conclusion: 1 The pharmacological activity of the protective effects of Gigantol on the retina of DR mice may be related to the TNF-?/NF-?B pathway,and reflect a dose-dependent relationship.2 The anti-inflammatory mechanism of Gigantol may be related to the inhibition of NF-?B transcriptional activity.3 Gigantol has a function similar to the TNF-?/I?B?/NF-?B signaling pathway inhibitor,which can attenuate high glucose-induced inflammatory damage by interfering with TNF-?/I?B?/NF-?B signaling pathway.