Protective Effects Of Astragaloside Ⅳ On High Glucose-induced Human Mesangial Cell Injury And Its Mechanisms

ObjectiveTo investigate the protective effects of Astragalosides IV(As-IV) on highglucose-induced Human mesangial cell (HMC) injury in vitro and further explore thepathogenesis of diabetic nephropathy(DN) and the molecular mechanism of As-IV inreducing DN, in order to provide theoretical basis for its clinical application.Methods1. HMC was cultured with low glucose DMEM medium containing10%fetal bovineserum, and incubated at37℃with5%carbon dioxide. Cells at logarithmic growthphase were taken for the experiment.2. The cultured HMC was randomly divided into9groups and treated as follows:normal glucose group (NG), Mannitol group (MA, osmolarity control), high glucosegroup (HG), Tempol group, SB431542group, and different doses of As-IV groups.NG group were cultured with low glucose culture medium(5.5mmol/L D-glocose), HGgroup were cultured with high glucose culture medium (30mmol/L D-glocose), MAgroup were cultured with low glucose culture medium (24.5mmol/L Mannitol),Similarly, Tempol group were cultured with high glucose culture medium (100μmol/LTempol), SB431542group were cultured with high glucose culture medium (10μmol/LSB431542), different doses of As-IV groups were cultured with high glucose culturemedium (As-IV12.5，25，50，100μmol/L). Also，different doses of As-IV groups were cultured with low glucose culture medium (As-IV12.5，25，50，100μmol/L) were setup. After treatment for48hours, the growth status of HMC was observed under invertedmicroscope, and the proliferation of HMC was analyzed by using MTT assay. From theinitial results, effective concentrations were chosen for the next experiment; howeverthe effect of osmolarity on HMC was excluded.3. In a subsequent experiment cells at logarithmic growth phase were randomly dividedinto7groups: NG group, HG group, Tempol group, SB431542group and As-IV groups(25，50，100μmol/L). After treatment for48hours, the levels of collagen type Ⅳ (ColⅣ), matrix metalloproteinase-9(MMP-9) and tissue inhibitor of metalloproteinase-1(TIMP-1) were detected from cell lysates by using enzyme-linked immunosorbent assay(ELISA).4. Also the content of reactive oxygen species (ROS) was measured by using2,7-dichlorodihydro fluorescin diacetate (DCFH-DA). Additionally, the expression oftransforming growth factor β1(TGF-β1), p-Smad2/3, NADPH oxidase4(NOX4) andcanonical transient receptor potential6(TRPC6) were analyzed by using Western blot.Results1. From the microscopic studies，it was observed that HMC in HG group increased innumber and cell volume during a period of48hours compared with the NGgroup,which implys that HMC was injured by the high glucose.The cell hypertrophyinduced by high glucose was inhibited by Tempol, SB431542and As-IV（12.5，25，50，100μmol/L)，which implys that As-IV has protective effects on high glucose-inducedHMC injury.2. Similarly from the MTT assay results, HG could increase the proliferation of HMCduring a period of48hours compared with the NG group (P<0.01). Compared with HG group, Tempol, SB431542, and As-IV（12.5，25，50，100μmol/L）groups significantly(P<0.01or P<0.05) inhibited HMC proliferation. As-IV inhibited HMC proliferation ina concentration-dependent manner. As-IV had no obviously effect on HMC culturedwith low glucose. And there was no significant difference between the normal groupand the MA group.3. The levels of Col Ⅳ, TIMP-1and MMP-9increased in the HG incubated HMCgroup but the ratio of MMP-9/TIMP-1was low. Tempol, SB431542, and As-IV（25，50，100μmol/L）groups could improve abnormal secretion of cytokines induced by highglucose medium and the effect of As-IV was in a concentration-dependent manner,implying that As-IV can regulate the synthesis and degradation of ECM in HMC.4. The mean DCF fluorescence intensity of HMC cultured in high-glucose mediumincreased compared with the NG group, which implies that the level of intracellularROS in HG group increased. Both Tempol and SB532542could reduce the mean DCFfluorescence intensity of HMC induced by high glucose medium, implying that theycould suppress the production of intracellular ROS. The level of intracellular ROS inhigh glucose medium was also inhibited by As-IV in a concentration-dependent manner,implying that As-IV can suppress the oxidative stress induced by high glucose.5. Western blot analysis results show that, compared with the NG group, the expressionof TGF-β1, p-Smad2/3and NOX4proteins were increased in HG group，while theexpression of TRPC6protein was significantly decreased(P<0.01). Also, Tempol,SB431542and As-IV（25，50，100μmol/L）treatments decreased expression of TGF-β1,p-Smad2/3, and NOX4, but significantly increased expression of TRPC6in HMCcultured in high glucose medium. The most effective concentration of As-IV was100μmol/L. ConclusionAs-IV has protective effects on high glucose-induced HMC injury. The possibleunderlying mechanisms may be through inhibition of HMC proliferation, andsuppression of oxidative stress and abnormal secretion of cytokines induced by highglucose; decreasing the expression of TGF-β1and p-Smad2/3protein, and increasing theexpression of TRPC6protein.