| Objective: The effect of Huang Qi compound granules(HQCGs)on senescence of Rat mesangial cells(RMCs)induced by high glucose was observed in vitro,and the mechanism of anti-senescence of Rat Mesangial cells(RMCs)induced by high glucose was discussed.Materials and methods: Serum containing HQCGs was prepared according to serum pharmacology.CCK-8 assay was used to detect the proliferation activity of RMCs.RMCs senescence were evaluated according to cell morphology,positive rate of SA-β-gal staining and cell cycle distribution.The level of ROS in RMCs was measured using the fluorescent probe DCFH-DA.Western blot was used to detect the expression of Sirt1/Foxo1 signal transduction pathway related proteins.After Sirt1 specific inhibitor EX527 was added to inhibit the expression of Sirt1 protein,the expression of related proteins,the positive rate of SA-β-gal staining,and the level of ROS were detected.Results:1.Cell activity was detected by CCK-8 methodIn the normal group,the cell proliferation activity increased with time(P<0.01).In high glucose group,cell proliferation activity decreased with time(P<0.01).At 24 h,compared with the normal group,the proliferation activity of RMCs cultured in vitro under high glucose was significantly increased(P<0.01).From 48 h to 72 h,compared with the normal group,the proliferation activity of the high glucose group decreased with time(P<0.01).At24 h,the proliferation activity of HQCGs group was significantly lower than that of the high glucose group(P<0.01),and there was no significant difference between the HQCGS group and the normal group(P>0.05).Compared with the high glucose group,the cell viability of the HQCGs group increased significantly(P<0.01),showing a time-dependent manner from48 h to 72 h.At the time point of 48 h,compared with the high glucose group,the cell proliferation activity of the low and medium dose groups of HQCGs was increased in a concentration-dependent manner(P<0.01).There was no significant difference in cell proliferation between the high dose group and the high glucose group(P>0.05).2.Morphological changes of cellsNormal RMCs are spindle-shaped or star-shaped,stereoscopic,mononuclear,and well-defined.Aging RMCs increased in volume,lost original shape,flattened,increased intracellular particles,increased dikaryotic and polykaryotic cells,and unclear boundaries.At24 h,there was no significant difference in cell morphology among all groups.From 48 h to72h,compared with the normal group,the cells in the high glucose group increased in size,lost their original shape,flattened cells,increased intracellular particles,more bi-conuclear and polykaryotic cells,and unclear boundaries.The above aging characteristics increased with the extension of time.After the intervention of HQCGs drug-containing se-rum,the volume of cells was reduced and returned to spindle or star shape with stereoscopic morphology.The intracellular granules were reduced,monocytosis was reduced,and the boundaries of double and polykaryotic cells were reduced.The effect of different concen-trations of HQCGs on morphological changes of cells was observed at 48 h time point when the cell state was more stable and controllable.It was found that compared with the high-glucose group,the cell morphology of low-dose and medium-dose groups tended to be normal.There was no significant difference in cell morphology between high dose group and high glucose group.3.Positive rate of SA-β-gal stainingThe expression of β-gal in the normal group was low,but the number of positive cells increased slowly with time(P<0.05).The positive rate of cell staining in high glucose group increased significantly with time(P<0.01).At 24 h,there was no significant difference in the positive rate of cell senescence staining among all groups(P>0.05).From 48 h to 72 h,compared with the normal group,the positive rate of senescence staining in the high glucose group increased significantly with time(P<0.01).Compared with the high glucose group,the HQCGs group had a significant reduction in cell senescence staining at 48h-72h(P<0.01),and the effect was the best at 48 h.At 48 h,compared with the high glucose group,the positive rate of cell staining in the low and medium dose groups of HQCGs decreased significantly with the increase of the concentration of HQCGs(P<0.05).There was no significant difference in the positive rate of cell staining between the high-dose group and the high glucose group(P>0.05).4.Cell cycle distributionAt 48 h,compared with the normal group,the number of cells arrested in G0/G1 phase in the high glucose group was significantly increased,and the number of cells in S phase and G2/M phase was significantly decreased(P<0.01).Compared with the high glucose group,the percentage of cells in G0/G1 phase in the low and medium dose groups decreased significantly,and the percentage of cells in S phase and G2/M phase increased significantly in a concentration-dependent manner(P<0.01).Compared with the high glucose group,there was no significant difference in G0/G1 phase and S phase cells in the high dose group(P>0.05).5.DCFH-DA was used to detect intracellular ROS levelsIt was found that the intracellular ROS level in the normal group increased slowly with time(P<0.01).ROS levels in RMCs treated with high glucose for 24 h,48h and 72 h were significantly increased with time(P<0.01).Compared with the high glucose group,ROS levels in RMCs treated with HQCGs for 24 h and 72 h decreased(P<0.05).At 48 h,intracellular ROS levels were significantly decreased in the HQCGs group.At 48 h,compared with the high glucose group,the ROS levels in the low and medium dose groups of HQCGs decreased with the increase of the concentration of HQCGs(P<0.05).The level of ROS in the high dose group was higher than that in the high glucose group(P<0.01).6.The changes of Sirt1/Foxo1 signaling pathway were detected by Western blot48h and medium dose of HQCGs were selected as the optimal action time and concentration to detect the expression of Sirt1/Foxo1 signal transduction pathway related proteins.Compared with the normal group,the expression of Sirt1 and Foxo1 protein in the high glucose group decreased(P<0.01).Compared with the high glucose group,the protein expressions of Sirt1 and Foxo1 in the medium-dose group of HQCGs were increased(P<0.05).7.Sirt1 specific inhibitor EX527 was addedTo further verify the relationship between the effect of HQCGs on RMCs and Sirt1/Foxo1 signal transduction pathway,Sirt1 specific inhibitor EX527 was added to inhibit the expression of Sirt1 protein.Compared with those in medium dose group of HQCGs,the protein expression levels of Sirt1 and Foxo1 of cells in medium dose group of HQCGs+100nmol/L EX527 group were significantly decreased(P<0.05),the positive rate of SA-β-gal staining was significantly increased(P<0.01),and the intracellular ROS level increased(P<0.01).Conclusion:1.High glucose can accelerate the senescence process of RMCs cultured in vitro,and the degree of cell senescence is aggravated with the extension of time.2.HQCGs can delay RMCs senescence.3.The anti-aging effect of HQCGs on RMCs induced by high glucose may be achieved by up-regulating the expression of Sirt1/Foxo1 pathway related proteins and reducing oxidative stress. |
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