The Mechanism of Sirt3 Mediated Mitochondrial Autophagy Inhibition on Nlrp3 Inflammasome Activation in Human Retinal Capillary Endothelial Cells Under High Glucose Condition

Abstract

To explore the regulatory effect of Sirt3 on mitochondrial stress, autophagy imbalance and inflammation in human retinal capillary endothelial cells (HRCECs) induced by high glucose. HRCECs were used for experimental studies. According to the research plan, the cultured cells were divided into the following groups: control group (HRCECs cultured under normal conditions), hypertonic group (dissolve 546 mg mannitol in 100 mL of complete culture medium, and prepare a 30 mmol/L stock solution), high glucose group (dissolve 540 mg D-glucose anhydrously in 100 mL complete culture medium, and prepare a 30 mmol/L stock solution). High-glucose+LV-vector group, high-glucose+LV-Sirt3 group, highglucose+ mdivi-1 group, high-glucose+LV-vector+mdivi-1 group and high-glucose+LV-Sirt3+mdivi-1 group. Apoptosis was analyzed by flow cytometry. Cellular ROS levels were measured by flow cytometry. ELISA detection kits were used to detect the levels of IL-18 and IL-1β in cultured cells. Cellular LC3, SIRT3, P62 and NLRP3 expression was analyzed by immunofluorescence. The protein expression of SIRT3, LC3-I, LC3-II, P62 and NLRP3 was analyzed by Western blotting. The mitochondrial membrane potential of cells was measured using JC-1 staining based on flow cytometry. The cell apoptosis rate in the hypertonic group was higher than that in the normal group (P <0.05), and the cell apoptosis rate in the high-glucose group was higher than that in the hypertonic group (P <0.05). The levels of IL-18 and IL-1β in the high glucose group were higher than those in the normal group and the hypertonic group (P <0.05). There was no difference in the levels of IL-18 and IL-1β between the hypertonic group and the normal group (P >0.05). The expression of LC3 and SIRT3 in the high glucose group was lower than that in the normal group and hypertonic group (P < 0>05). The protein expression of SIRT3 and LC3-II/LC3-I in the high-glucose group was lower than that of the normal group and the hypertonic group (P <0.05). The protein expression of P62 and NLRP3 in the high-glucose group was higher than that of the control group (P <0.05). In high-glucose group and high-glucose+LV-vector group the levels of IL-18 and IL-1β were higher than those in the normal group (P <0.05), and the levels of IL-18 and IL-1β in the high-glucose+LV-Sirt3 group were lower than those in the high-glucose+LV-vector group. (P <0.05). The mitochondrial membrane potential of the high-glucose group and the high-glucose+LV-vector group was lower than that of the normal group (P <0.05). The mitochondrialmembrane potential of the high-glucose+LV-Sirt3 group was higher than that of the high-glucose+LV-vector and high-glucose groups. The expression of NLRP3 and P62 in the high-glucose group and the high-glucose+LV-vector group was higher than that in the normal group (P <0.05). The expression of NLRP3 and P62 in the high-glucose+LVSirt3 group was lower than that in the high-glucose+LV-vector group. The cell apoptosis rate in the high-glucose group and the high-glucose+LV-vector group was higher than that in the normal group (P < 0.05). The cell apoptosis rate in the high-glucose+LV-Sirt3 group The rate was lower in the higher sugar+LV-vector group (P <0.05). The protein expressions of SIRT3 and LC3-II/LC3-I in the high-glucose group and the high-glucose+LV-vector group were lower than those in the normal group (P <0.05). High-glucose+LV-Sirt3 group SIRT3 and LC3-II/LC3-I protein expression increased than the high-glucose group and high glucose+LV-vector group was increased compared with the normal group (P <0.05), and the protein expression of P62 and NLRP3 in the high-glucose+LV-Sirt3 group was decreased (P <0.05) than in the high-glucose group and the high-glucose+LV-vector group. High glucose+mdivi-1 The levels of IL-18 and IL-1β in the high-glucose+LV-vector+mdivi-1 group were higher than those in the high-glucose group (P <0.05). The levels of IL-18 and IL-1β in the high-glucose+LV-Sirt3+mdivi-1 group decreased than in the high-glucose group, high-glucose+mdivi-1 group and high-glucose+LV-vector+mdivi-1 group (P <0.05). The expression of NLRP3 and P62 protein in the highglucose+ mdivi-1 group and high-glucose+LV-vector+mdivi-1 group increased than in the high-glucose group (P <0.05), and the expression of NLRP3 and P62 proteins in the high-glucose+LV-Sirt3+mdivi-1 group decreased (P <0.05). The ROS level of cells in the high-glucose+mdivi-1 group and in the high-glucose+LV-vector+mdivi-1 group was higher than that in the high-glucose group (P < 0.05). The ROS level of cells in the high-glucose+LV-Sirt3+mdivi-1 group was decreased (P <0.05) than that in the high-glucose+LV-vector+mdivi-1 group. The cell apoptosis rate in the highglucose+ mdivi-1 group and the high-glucose+LV-vector+mdivi-1 group was higher than in the high-glucose group (P < 0.05). The apoptosis rate of the high-glucose+LV-Sirt3+mdivi-1 group was lower than that of the high-glucose+mdivi-1 group (P <0.05). The protein expression of SIRT3, LC3-I/LC3-II, P62 and NLRP3 was analyzed by Western blotting. The protein expression of SIRT3 in the high-glucose group was lower than that in the high-glucose+mdivi-1 group and the high-glucose+LV-vector+mdivi-1 group (P <0.05), the expression of SIRT3 protein in the high glucose+LV-Sirt3+mdivi- 1 group was higher than that in the high-glucose+mdivi-1 group (P <0.05). The protein expression of LC3-I/LC3-II in the high-glucose group was higher than that in the high-glucose+mdivi-1 group and the high-glucose+LV-vector+mdivi-1 group (P <0.05). The protein expression of LC3-I/LC3-II in the high-glucose+LV-Sirt3+mdivi-1group was higher than that in the high-glucose+mdivi-1 group (P <0.05). The protein expression of P62 and NLRP3 in the high-glucose group was lower than that in the high-glucose+mdivi-1 group and the high-glucose+LV-vector+mdivi-1 group (P <0.05). The protein expression of high-glucose+LV-Sirt3+mdivi-1 group was lower than in the high-glucose+mdivi-1 group (P <0.05). Sirt3 effectively regulates inflammatory cell apoptosis in human retinal capillary endothelial cells by alleviating mitochondrial stress and autophagy imbalance under high glucose environment. Overexpression of Sirt3 reduces cell apoptosis rate and inflammatory response, stabilizes mitochondrial membrane potential, and reduces ROS production, thereby playing a key protective role in high glucose-induced cell damage.