Study On The Mechanism Of Tang Wang Ⅱ In Regulating HMGB1/TLR4/NF-κB Signaling Pathway In Diabetic Retinopathy

Diabetic retinopathy is a common microvascular complication of diabetes with complex pathological mechanisms.As an important organ that regulates the metabolism of sugars,lipids,proteins and other substances,the liver is the main organ of insulin action and an important site of insulin resistance.The liver is crucial to the development and progression of diabetes and its complications.Hyperglycaemia causes abnormalities in the hepatic oxidative stress system,inducing hepatocyte damage and contributing to chronic inflammation,insulin resistance and fatty acid oxidative stress.Chronic low-level inflammation plays an important role in the pathogenesis of DR.In our group’s previous in vivo study,we found that the hepatologically-based formula Tang Wang Ⅱ had a protective effect on the liver and retina of rats with diabetic retinopathy.This study will investigate the mechanism of Tang Wang II in the treatment of diabetic retinopathy from the perspective of molecular biology,and provide a basis for later clinical studies.Objective:1.To analyze and screen the active ingredients and possible targets and mechanisms of Tang Wang Ⅱ in the treatment of diabetic retinopathy using a network pharmacology approach,and to provide a reliable methodological basis for subsequent cellular experiments.2.Establish a model of human retinal pigment epithelial cells(RPE)under high glucose,to observe the effect of Tang Wang II on the activity and apoptosis of human retinal pigment epithelial cells under high glucose.To investigate the regulatory mechanism of Tang Wang Ⅱ in improving diabetic retinopathy through HMGB1/TLR4/NF-κB signalling pathway,providing a molecular biological basis for later clinical application.3.To explore the relationship between miR-142-3P and HMGB1 expression in the serum of diabetic retinopathy patients using healthy subjects as controls,and to explore the potential biological markers of DR in diabetic patients from the perspective of genes.Methods:1.Network pharmacology study:Molecular database of Tang Wang Ⅱ was constructed through Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)from the active ingredients of drugs.Target datasets were built from The OMIM,GeneCards,and TTD databases with unified the gene names of UniProt database.The two datasets were mapped and processed to screen for duplicate targets.A PPI network of TCM components-disease targets was constructed using STRING online software.The core targets were screened by Cytoscape software network analysis.Finally,KEGG and GO enrichment analyses were performed using Metascape.2.Experimental study methods.(1)ARPE-19 was treated with different concentration gradients of glucose DMEM/F12 complete culture medium,cultured for 24h and 48h.Then cell activity was detected by CCK8 to screen the glucose concentration and culture time for mouldmaking.(2)different concentration gradients of drug-containing serum for SD rats with Tang Wang Ⅱ was prepared,and cultured ARPE-19 cells under high glucose for 24h.The effect of Tang Wang Ⅱ drug-containing serum on the activity of ARPE-19 cells under high glucose was detected by the CCK8.(3)Establish a model of ARPE-19 cells under high glucose,conditioned culture medium was prepared according to normal group,model group,blank control group,and low dose,medium dose and high dose groups of Tang Wang Ⅱ to interfere with normal glucose and high glucose concentration of ARPE-19 cells.Hoechst-PI staining were used to detect the effect of Tang Wang Ⅱ on the apoptosis of ARPE-19 cells under high glucose.(4)Establish a model of ARPE-19 cells under high glucose,conditioned cultures were prepared according to normal group,model group,mannitol control group,blank control group,and low dose,medium dose and high dose groups of Tang Wang Ⅱ to interfere with normal glucose and high glucose concentration of ARPE-19 cells.The expression of HMGB1,TLR4,NF-κB and IL-1β in each group was detected by qPCR,the expression levels of HMGB1,TLR4,NF-κB and p-NF-κB proteins in the cells were detected by Western blot,the results of IL-1β,IL-6 and TNF-α expression in the cell culture supernatant of each group were detected by ELISA,immunofluorescence and semiquantitative analysis were used to detect the expression of HMGB1、TLR4、NF-κB and the location of proteins.Final qPCR assay to detect the expression level of miR-142-3P in drug-containing serum.3.Clinical study method:A cross-sectional study was conducted by collecting 30 healthy subjects,30 DM subjects,26 NPDR subjects and 25 PDR subjects according to the inclusion and exclusion criteria.The expression level of miR-142-3P in serum was detected by qPCR and the levels of HMGB 1,TNF-α,IL-6 and IL-1β in serum was detected by ELISA.Results:1.The results of the network pharmacology study showed that the main biological processes involved in the treatment of diabetic retinopathy with Tang Wang Ⅱ are inflammation,immunity and apoptosis,as well as the AGE-RAGE signaling pathway,PI3K-Akt signaling pathway,MAPK signaling pathway,IL-17,TNF and other inflammatory signaling pathways.2.Experimental studies(1)ARPE-19 cells were cultured in complete medium with different concentrations of glucose(5,25,35,45,55 and 65 mmol/L),and the cells showed a growth-promoting tendency when the glucose concentration was less than 25 mmol/L,and an inhibitory tendency when the glucose concentration was greater than 25 mmol/L,where the cells showed a growth-promoting tendency when the glucose dose was 55 mmol/L and The cell viability was significantly inhibited when the glucose dose was 55mmol/L and 65mmol/L(P<0.05).there was no statistically significant difference between the cell survival rate at 24h and 48h(P>0.05).(2)Compared with the normal group of cells cultured in low sugar complete medium,the inhibition rate of ARPE-19 cells in each group was not statistically significant(P>0.05)when compared with the normal group of cells cultured in 5%and 10%volume fractions of the drug-containing serum.The difference in the inhibition rate of ARPE-19 cells in each group was statistically significant(P<0.05)when comparing the 20%and 30%volume fractions of the drug-containing serum with the normal group.(3)The results of Hoechst-PI staining showed that after PI staining,the model group was able to observe a rosy red colour with typical apoptotic features such as chromatin fixation and nuclear fragmentation,which were particularly obvious,while the drug-containing serum group showed between the two groups above.The apoptosis rates of all drug-containing serum groups were significantly lower than those of the model group(P<0.05),indicating that the apoptosis of ARPE-19 cells under high glucose was inhibited by the drug-containing serum of Tang Wang Ⅱ,and the inhibition was most obvious in the medium-dose group,and the difference was statistically significant(P<0.05).The change of apoptosis rate was not significant in the blank group compared with the model group(P>0.05).(4)a.The results of qPCR assay showed that the relative expression of HMGB1,TLR4,NF-κB and IL-1β mRNA in the model group was significantly higher than that in the normal group(P<0.05),the difference between the normal group and the mannitol group was not statistically significant(P>0.05),and the difference between the model group and the blank control group was not statistically significant(P>0.05).b.Western blot results showed that the relative expressions of HMGB1,TLR4,pNF-κB and NF-κB in the model group were significantly higher than those in the normal group,using GAPDH as the internal reference and the normal group as the reference(P<0.05);the relative expressions in the low,medium and high dose groups were between the model and normal groups(P<0.05),indicating that the intervention of Tang Wang Ⅱ in ARPE-19 cells under high glucose was effective.c.ELISA results showed that the levels of inflammatory factors in the cell culture supernatant of each group were the lowest in the normal group and the highest in the model group,and the differences between them were statistically significant(P<0.05).and the difference was not statistically significant(P>0.05)in the mannitol control group compared with the normal group.The Chinese medicine intervention group was between the normal group and the model group,and the difference between them and the model group was statistically significant(P<0.05).d.Immunofluorescence staining and semi-quantitative analysis showed that HMGB1 was localized in the nuclei of ARPE-19 cells in the low glycemic control group.In the model group,HMGB1 protein expression was upregulated in ARPE-19 cells,and HMGB1 was transferred from the nucleus to the cytoplasm.The cytoplasmic HMGBA expression in the serum-containing group was between that in the normal control group and the model group,with the most significant improvement in cytoplasmic HMGBA expression in the mid-dose group compared to the model group.The increased expression of NF-κB protein in ARPE-19 cells and the appearance of NF-κB in the nucleus of the model group indicated that high glucose prompted the transfer of NF-κB protein from the cytoplasm to the nucleus of ARPE-19 cells to complete the synthesis and secretion of downstream inflammatory factors.The NF-κB expression in the drug-containing serum group was reduced to different degrees compared to the model group.e.The results of qPCR of drug-containing serum showed that the expression of miR-142-3P in the drug-containing sera was compared with that of the blank control group.The results showed that the expression of miR-142-3P in the drug-containing serum group was higher than that in the blank control group,in which the expression in the medium and high dose groups was significantly higher than that in the blank control group(P<0.05).3.Clinical study:HMGB1,TNF-α and IL-1β were higher in PDR patients than in NPDR patients,and the difference was statistically significant(P<0.05).The relative expression of serum miR-142-3P was lower in the PDR group than in the NPDR group,and the difference was statistically significant(P<0.05).Pearson correlation analysis showed that serum miR-142-3P was negatively correlated with HMGB1,TNF-α,and IL-1β levels in DR patients(P<0.05).Conclusion:1.The network pharmacology analysis revealed numerous targets associated with diabetic retinopathy,and the enrichment analysis revealed that Tang Wang II treatment of diabetic retinopathy was associated with immune and inflammatory apoptotic pathways,providing a methodological basis for further study of the molecular mechanisms associated with Tang Wang Ⅱ.2.High glucose-induced ARPE-19 cells can inhibit cell activity,cause apoptosis,and promote the expression of HMGB1,TLR4,NF-κB and downstream inflammatory factors,and destroy the blood-retinal external barrier.3.Tang Wang Ⅱ May inhibit the expression of HMGB1/TLR4/NF-κB pathway protein in ARPE 19 cells under high glucose conditions by up-regulating the expression of miR-142-3p,improve inflammatory response,reduce the damage of blood-retinal external barrier,and play a role in treating DR.4.Serum miR-142-3p/HMGB1 is associated with the degree of diabetic retinopathy,and miR-142-3p may be a potential biological marker of the occurrence of DR in diabetic patients.