| BackgroundDiabetic retinopathy(DR)is a serious microcirculation disorder caused by glucose metabolism disorder,and it is also one of the most common chronic complications of diabetes.The pathogenesis of DR involves a huge regulatory network.Recent studies have found that chronic low-grade inflammation is one of the important pathophysiological mechanisms of diabetic retinopathy.Long-term chronic low-grade inflammation may also cause the abnormal immune system,and break the balance of immune microenvironment,which results in the occurrence of disease.Hence,more and more inflammatory factors have attracted people’s attention.As an anti-inflammatory factor,interleukin-38(IL-38)is a newly discovered member of the IL-1 family cytokines in recent years.At present,many studies have shown that IL-38 is closely related to various autoimmune diseases such as ankylosing spondylodiscitis,cardiovascular diseases,and inflammatory bowel disease.Studies have shown that in a rat model of oxygen-induced retinopathy,intraperitoneal injection of IL-38 can inhibit the formation of retinal neovascularization.Moreover,administration of IL-38 can reduce the proliferation of vascular endothelial cells and the formation of vascular cavity induced by vascular endothelial growth factor(VEGF).In the alkali-induced corneal neovascularization mouse model,topical application of IL-38 can inhibit the formation of corneal neovascularization.Therefore,IL-38 may play an important role in the occurrence and development of diabetic retinopathy by participating in the inflammatory response and the process of neovascularization.ObjectiveIn this study,the expression level of IL-38 in serum and aqueous humor of DR patients was separately detected.Moreover,the effect of IL-38 on the expression of inflammatory factors and pro-angiogenesis produced by human-retinal pigment epithelial cell(ARPE-19)under high glucose environment was studied in vitro to preliminarily explore the potential role of IL-38 in the development of DR.The possible related molecular biological mechanisms are expected to deepen the understanding of the pathogenesis of DR and provide new ideas for the diagnosis and treatment of DR.MethodsThirty-two PDR patients(32 eyes)who were hospitalized in Department of Ophthalmology,the First Affiliated Hospital of Zhengzhou University from January 2020 to December 2020 were included,including 13 males and 19 females.The average age of PDR patients was(58.53±5.03)years.The control group included 30 patients(30 eyes)with idiopathic macular hole without diabetes who were treated in our department during the same period,including 9 males and 21 females.The average age of control group was(60.23±4.64)years.On the next morning after the informed consent form was signed for specimen collection,2mL of blood from the median cubital vein was drawn,and after centrifugation,0.5mL of the supernatant was taken and transferred to a refrigerator at-80℃.Aqueous humor was collected when PDR patients received intravitreal injection of anti-VEGF drugs and the control group underwent standard three-channel vitrectomy.Under the microscope,about 80-100 μL of undiluted aqueous humor is slowly extracted from a 0.33mm needle.The aqueous humor samples were immediately transferred to a refrigerator at-80℃ for storage.The expression level of IL-38 in the serum and aqueous humor of the PDR group and the control group were determined by enzyme-linked immunoassay(ELISA).ARPE-19 was cultured in vitro and divided into low sugar group(5.5 mmol/L glucose),mannitol control group(5.5 mmol/L glucose+19.5 mmol/L mannitol),high glucose group(25 mmol/L glucose),high glucose+IL-38 group(25 mmol/L glucose+5 ng/mL IL-38).Under different culture conditions,quantitative PCR was used to detect the mRNA expression of IL-6,tumor necrosis factor-a(TNF-α),and hypoxia induced factor-1α(hypoxia induced factor-1α)in RPE cells.The ELISA method was used to detect the secretion changes of IL-6,TNF-α,and VEGF in the supernatant of RPE cells,and the protein expression changes of HIF-la in RPE cells were detected by Western blot.The SPSS 23.0 software was used to analyze the relevant data.For quantitative data,the independent sample t test was used for the comparison of the two groups of data,one-way analysis of variance was used for multiple comparisons,and Tukey test was used for comparison of data between two groups.And the difference of qualitative data was compared by chi-square test.P<0.05 was considered as statistically significant.Results1.The concentration of IL-38 in serum of the PDR group(152.07±38.19 pg/mL)was higher than that of the control group(121.23±29.99 pg/mL),and the difference was statistically significant(P<0.05).2.The concentration of IL-38 in aqueous humor of the PDR group(44.23±8.45 pg/mL)was higher than that of the control group(35.29±7.23 pg/mL),and the difference was statistically significant(P<0.05).3.Quantitative PCR results showed that compared with the low glucose group,the mRNA expression of IL-6,TNF-α,and HIF-1α was statistically higher in the high glucose group(P<0.05).In addition,the mRNA expression of IL-6,TNF-α,and HIF1α in the high glucose+IL-38 group was statistically lower than that in the high glucose group,but higher than that in the low glucose group(P<0.05).4.ELISA results showed that compared with the low glucose group,the secretion level of IL-6,TNF-α,and VEGF was statistically higher in the high glucose group(P<0.05).In addition,the secretion level of IL-6,TNF-α,and VEGF in the high glucose+IL-38 treatment group was statistically lower than that in the high glucose group,but higher than that in the low glucose group(P<0.05).5.Western blot results showed that compared with the low glucose group,the protein expression of HIF-la was increased in the high glucose environment,and the difference was statistically significant(P<0.05).In addition,the protein expression of HIF-1α in the high glucose+IL-38 group was statistically lower than that in the high glucose group,but higher than that in the low glucose group(P<0.05).Conclusions1.The expression levels of IL-38 in the serum and aqueous humor of PDR patients were increased,suggesting that IL-38 may be involved in the development of DR.2.IL-38 can reduce the mRNA and protein expression levels of IL-6 and TNF-αin ARPE cells under hyperglycemia condition.The results indicate that IL-38 may play a role in the inflammation in the eye by regulating the expression of IL-6 and TNF-α,and then participates in the development of DR.3.IL-38 can reduce the secretion of VEGF,the mRNA and protein expression levels of HIF-1α in ARPE cells under hyperglycemia.The results indicate that IL-38 may down-regulate the expression of VEGF by inhibiting the expression of HIF-1α and then participates in the development of DR. |
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