The Effect Of Gambogic Acid On Retinal Inflammation In Diabetic Mice Fed With High-fat Diet And The Underlying Mechanism

Objective: Diabetic Retinopathy(DR)is an ocular microvascular complication that seriously threatens vision in people with diabetes.In the early stage of DR,the occurrence and development of DR are closely related to systemic and retinal inflammation and immune response.In vitro and in vivo studies have proved that gambogic acid(GA),one of the main active monomers of gambogic,has anti-inflammatory effect on DR with "lipotoxicity" stimulation.Methods: This study can be divided into three parts: first,we used high glucose(HG)combined with palmitic acid to establish a cell model of RPE inflammatory response induced by glycolipid toxicity in human retinal pigment epithelial cells(ARPE-19).Cell viability was detected by CCK8,and the expression levels of IL-1? and TNF-? were detected by ELISA.Western blot was used to detect the expression of NLRP3 inflammasome related components,including TXNIP?pro-caspase-1/cleaved-caspase-1?ASC?pro-IL-1? /cleaved-IL-1??IL-18 and Nrf2/Nrf2(nucleus)protein.Then,GA was used to intervene the cell model,apoptosis was detected by Annexin V-FITC;levels of inflammatory cytokines were detected by ELISA;the expression of NLRP3 inflammasome components and Nrf2 signaling pathway including its downstream products:heme oxygenase-1(HO-1)?NADPH: quinone oxidoreductase 1(NQO1)were detected by Western blot;The expression of Nrf2 in ARPE-19 was detected by immune-fluorescence staining.The inhibition experiment of Nrf2 si RNA verified that the anti-inflammatory effect of GA was regulated by Nrf2 pathway.Finally,the corresponding in vivo study further confirmed the inhibitory effect of GA on DR inflammatory response.The mouse model of early DR was established by high fat feeding combined with STZ intraperitoneal injection,C57BL/6 mice were randomly divided into 6 groups: control group,high-fat feeding group,high-fat feeding + STZ model group and high glucose and high-fat model with different doses of GA treatment group;the control group received normal diet,the high-fat group was given high-fat diet,the model group and the treatment group were given high-fat feeding combined with STZ intraperitoneal injection to establish the model,and then the treatment group was given different doses of GA treatment.1.The levels of nonesterified fatty acid(NEFA)were detected by ELISA,the changes of retinal vascular permeability were detected by fundus fluorescein angiography(FFA),and the changes of retinal layer and morphology were detected by optical coherence tomography(OCT).2.After GA treatment for 4 weeks,HE staining and Masson staining were used to observe the morphological and fibrosis changes in retina.3.The localization and expression of NLRP3 and Nrf2 were detected by immunofluorescence staining and immunohistochemical analysis.4.The expression levels of NLRP3 inflammasome related components in mouse retina were detected by Western blot.5.Western blot and real-time PCR were used to detect the protein expression and m RNA levels of Nrf2?HO-1 and NQO1.Results:1.After treated with 200 ?m PA and HG for 24 h,CCK8 and ELISA results showed that the activity of ARPE–19 cells decreased,the contents of IL-1 ? and TNF-? increased,and the protein expression of NLRP3 and Nrf2 increased.2.Treated with 0.5 ?m?1 ?m and 2 ?m GA for 24 hours showed increased cell viability by CCK8 experiment;ELISA analysis showed that the levels of inflammatory cytokines IL-1? and TNF-? were significantly inhibited at the level of sub toxic cells in a dose-dependent manner with GA concentration;Western blot showed that GA could down regulate the expression of NLRP3 inflammatory body components and increase the protein expression of Nrf2 into the nucleus;immunofluorescence analysis showed that increased Nrf2;RT-q PCR showed that GA significantly increased the m RNA levels of HO-1 and NQO1,which were downstream Nrf2 transcription dependent.In addition,Nrf2 si RNA assay confirmed that after Nrf2 was silenced,NLRP3 inflammasome components and downstream cleaved-IL-1? were not affected by GA,and continued to increase,while Nrf2 protein level continued to decrease.3.The content of NEFA in mice fed with high-fat diet combined with STZ injection was significantly increased;FFA + OCT showed increased vascular permeability and unclear local retinal hierarchy;the expression level of NLRP3 inflammasome in retina of mice fed with high-fat diet combined with STZ injection was increased by Western blot;after GA treatment,1.5 mg/kg,3 mg/kg and 6 mg/kg GA decreased the expression of NLRP3 inflammasome and inhibited inflammatory cells in a dose-dependent manner He staining + Masson staining showed that GA could improve the structure of retina and reduce the fibrosis;at the same time,GA upregulated the protein expression and m RNA level of Nrf2,HO-1 and NQO1.In addition,immunofluorescence staining and immunohistochemistry analysis also confirmed that GA could reduce the expression of NLRP3 and increase the expression of Nrf2 in mouse retina.Conclusion: 1.ARPE-19 co-cultured with HG and PA can be used as a model of early inflammatory response of DR.2.GA can inhibit TXNIP/NLRP3/IL-1? by activating Nrf2/HO-1 signaling pathway.The results showed that the excessive activation of IL-1? signaling pathway could inhibit the inflammatory response of RPE cells induced by "glycolipid toxicity";the anti-inflammatory effect of GA on ARPE-19 depended on the activation of Nrf2 axis;3.High fat feeding combined with STZ injection mice showed the early changes of DR lesions under the stimulation of "glycolipid toxicity",the retina was in the inflammatory stress environment,and NLRP3 inflammatory body participated in the process of inflammation generation and inflammation cascade amplification.4.GA can activate Nrf2/HO-1 signal pathway of antioxidant axis,inhibit the over activation of TXNIP/NLRP3/IL-1?signal pathway,and plays an anti-inflammatory and protective role in early stage of retinal inflammation in diabetic mice.