| Diabetes macular edema(DME)is the main cause of visual impairment in diabetes retinopathy(DR),which can occur at any stage of DR.With the increasing number of patients with diabetes year by year,the incidence rate of DME is also on the rise.The pathogenesis of DME is complex and diverse,and is the result of the interaction of multiple pathogenic factors and multiple biological pathways.It is closely related to multiple inflammatory cytokines and metabolic pathways.Inflammation and oxidative stress seem to play a central role in this pathophysiology.Therefore,this experiment established a model of HRMEC injury induced by high glucose,selected PI3K/Akt and Nrf2/ARE signaling pathways closely related to inflammation and oxidative stress to conduct experiments,and explored the mechanism of Xiaozhong Granule in treating DME,providing a theoretical basis for clinical research.Objective1.To establish a high glucose model and observe the effect of Xiaozhong Granule containing serum on the morphology,apoptosis and ultrastructure of HRMEC cells.2.Based on PI3K/Akt and Nrf2/ARE signal pathway,the protective mechanism of Xiaozhong Granule on high glucose induced HRMEC was discussed.3.To observe the long-term efficacy and safety of Xiaozhong Granule and Conbercept in the treatment of DME.Methods1.Cell experiment:(1)Use CCK-8 detection method to screen the glucose concentration of high glucose injury model and detect cell viability.(2)The apoptosis morphology of HRMEC in each group was observed by TUNEL staining,and the apoptosis rate was detected by flow cytometry.(3)The ultrastructural changes of HRMEC(mitochondria)in each group were observed with biological transmission electron microscope.(4)Western blot was used to detect the protein expression of PI3K、AKT、p-PI3K、p-AKT、P65、p-P65,Bax,Bcl-2,Caspase-3,Nrf2,HO-1,SOD-1 in each group.(5)ELISA was used to detect the expression of TNF-α、IL-1β、IL-6 in each group.And the expression of ROS,MDA,GSH-Px in each group,which are indicators of oxidative stress.(6)RT-PCR was used to detect the expression of PI3K,AKT,Nrf2 and HO-1 mRNA in each group.(7)The expression of ROS in cells of each group was detected by immunofluorescence staining.2.Clinical trials:Through a randomized,controlled study,40 DME patients(40 eyes)were selected from September 2020 to March 2022,who were diagnosed as spleen and kidney yang deficiency and blood stasis blocking the eye meridian type.The test group was given Conbercept injection+Xiaozhong granules group(24 weeks),and the control group was given Conbercept injection+Chinese medicine placebo group(24 weeks).Both groups received a visit and evaluation every 4 weeks,and were followed up to 36 weeks and 48 weeks.BCVA,CMT,macular edema volume and TCM syndrome score of patients in the two groups were observed in January,March,June,September and December.Results1.Cell experiment:(1)CCK-8 detection method:HRMEC was cultured at different glucose concentrations(5mmol/L,25mmol/L,35mmol/L,45mmol/L,55mmol/L,65mmol/L).When the glucose concentration was less than 25mmol/L,the cells were in a proliferative state.When the concentration of glucose is greater than 25mmol/L,the cell growth is in a state of inhibition,and the higher the concentration of glucose,the worse the cell growth activity,in which the cell activity begins to decline significantly at 55mmol/L glucose,and the inhibition is the most significant.There was no significant difference in cell survival rate between 24h and 48h(P>0.05).Therefore,this experiment selected 55mmol/L glucose concentration to interfere with HRMEC for 24h to establish a high glucose injury model.(2)TUNEL staining:Compared with the normal group,the number of apoptotic cells in the model group increased significantly,with a statistical difference(P<0.05).The number of apoptotic cells in the model group and the blank control group was equal.Compared with the model group,the number of apoptotic cells in the drug-containing serum group decreased significantly(P<0.05).(3)Flow cytometry:The apoptosis rate of cells in the normal group was 3.48%,and that in the model group and the blank control group was 35.20%and 32.30%respectively.There was a statistical difference between the model group and the normal group(P<0.05).However,the apoptosis rate of cells in the serum containing drug group was 14.80%,which was significantly lower than that in the model group and the blank control group.There was a statistical difference between the groups(P<0.05).(4)Biological transmission electron microscope:Compared with the normal group,the model group had unclear nuclear boundary,swelling,disordered cytoplasmic structure,decreased mitochondrial number,dissolved mitochondrial membrane,broken inner ridge,and even vacuolar changes.Compared with the model group,the nucleus swelling of the drug-containing serum group was reduced,the number of mitochondria was increased,the inner ridge of mitochondria was slightly blurred,and the structure was basically complete.(5)Inflammatory factors:TNF-α,IL-1β and IL-6 in cells of each group.The expression of IL-6 in the model group was significantly higher than that in the normal group,and the difference was statistically significant(P<0.05).After intervention,the expression level of the drug-containing serum group was significantly lower than that of the model group(P<0.05).(6)Apoptosis protein:Compared with the normal group,the expression of Bax and Caspase-3 protein in the model group increased significantly,while the expression of Bcl-2 protein decreased significantly,and the difference was statistically significant(P<0.05).Compared with the model group,the expression of Bax and Caspase-3 Protein in the drug-containing serum group decreased significantly,while the expression of Bcl-2 protein increased significantly,and there was a statistical difference between the groups(P<0.05).(7)PI3K/Akt signal pathway related factors:The expression of PI3K mRNA and AKT mRNA in the model group decreased significantly,and there was a statistical difference between the two groups(P<0.05).Compared with the model group,the expression of PI3K mRNA and AKT mRNA in the drug-containing serum group increased significantly(P<0.05).Compared with the normal group,the expression of p-PI3K and p-AKT protein in the model group decreased,and the expression of p-P65 protein increased significantly(P<0.05).Compared with the model group,the expression of p-PI3K and p-AKT protein in the drug-containing serum group increased,while the expression of p-P65 protein decreased significantly(P<0.05).(8)Oxidative stress index:Compared with the normal group,the expression of ROS and MDA in the model group increased significantly,while the expression of GSH-Px decreased significantly,with significant statistical difference(P<0.05).Compared with the model group,the expression level of ROS and MDA in the drug-containing serum group decreased significantly,and the expression level of GSH-Px increased significantly(P<0.05).(9)Nrf2/ARE signal pathway related factors:Compared with the normal group,the expression of Nrf2 mRNA and HO-1 mRNA in the model group decreased significantly,with significant difference(P<0.05).Compared with the model group,the expression levels of Nrf2 mRNA and HO-1 mRNA in the serum group containing drugs were significantly higher(P<0.05).Compared with the normal group,the expression of Nrf2,HO-1 and SOD-1 in the model group decreased significantly(P<0.05).Compared with the model group,the expression of Nrf2,HO-1 and SOD-1 in the drug-containing serum group was significantly higher(P<0.05).(10)After the intervention of PI3K inhibitor:The expression of p-PI3K,p-Akt,p-Nrf2,HO-1 protein is as follows:compared with the normal control group,it is significantly reduced in the model group and inhibitor group.Compared with the model group and the inhibitor group,the expression in the drug-containing serum group and the drug-containing serum+inhibitor group increased,but the drug-containing serum+inhibitor group was between the model group and the drug-containing serum group,with statistical differences(P<0.05).The Protein p-PI3K/PI3K,p-Akt/Akt,p-Nrf2/Nrf2 ratio and HO-1 of each group were statistically different(P<0.05).2.Clinical trials:(1)Vision of two groups of patients:After treatment,the number of BCVA letters increased to varying degrees compared to baseline.The difference between the test group and the baseline at 1,3,6,9and 12 months after treatment was statistically significant(P<0.05).The difference between the control group and the baseline at 1,3,and 6 months after treatment was statistically significant(P<0.05).The difference between the two groups,after 1 month and 12 months of treatment,there was no significant difference(P>0.05).(2)CMT and macular edema volume of patients in two groups:After treatment,both CMT and macular edema volume decreased from baseline.One month after treatment,there was no statistically significant difference in CMT and macular edema volume between the test group and the control group(P>0.05).The CMT and macular edema volume of patients in the experimental group were significantly different from those in the control group at 3,6,9,and 12 months after treatment(P<0.05).(3)TCM syndrome score of two groups of patients:After treatment,the TCM syndrome scores have decreased to varying degrees.The TCM syndrome scores of the experimental group decreased significantly in both the 6th and 12th months of treatment compared to those before treatment,with a statistically significant difference(P<0.05).The difference between the treatment group and the control group was the most significant.In the control group,there was a statistically significant difference in TCM syndrome scores between the 12 months of treatment and before treatment(P<0.05).(4)Safety indicators:There was a statistically significant difference in HbAlc in the test group between 6 months of treatment and before treatment(P<0.05).During the treatment period,there were 2 patients with vitreous hemorrhage in the control group,and 1 patient with transient high intraocular pressure and 1 patient with vitreous hemorrhage in the test group.The incidence of adverse reactions in the two groups was 5%(2/40),with no statistically significant difference between the two groups(P>0.05).After 6 and 12 months of treatment,there were no significant abnormalities in liver and renal functions in both groups of patients,and no adverse reactions were observed in other patients.Conclusions1.Select 55mmol/L glucose concentration to interfere with HRMEC for 24h,and successfully establish a high glucose injury model.2.Xiaozhong Granule can improve the morphology of HRMEC cells induced by high glucose,inhibit cell apoptosis,and protect the integrity of mitochondrial structure.3.Xiaozhong Granule can improve the expression of inflammatory factors and apoptosis related proteins in HRMEC induced by high glucose.4.Xiaozhong Granule can activate The PI3K/AKT/NF-κB P65 signaling Pathway mediates inflammatory reactions and plays an anti-apoptotic role.5.Xiaozhong Granule can improve related indicators of oxidative stress,activate Nrf2/ARE signaling pathway,and play an antioxidant role.6.Xiaozhong Granule can play an anti-inflammatory,antioxidant,and anti-apoptotic role by activating the Nrf2/ARE pathway mediated by PI3K/Akt.7.It has been revealed that Xiaozhong Granule can protect the blood retinal barrier by multiple targets and pathways against high glucose induced HRMEC injury.8.Xiaozhong Granule combined with Conbercept Injection can improve the BCVA of DME patients,reduce CMT and macular edema volume,reduce the recurrence rate of macular edema,and improve the overall symptoms of DME Patients.The long-term efficacy is stable and safe. |
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