| Objective: The detection of serum containing Qingshen granules in low,medium and high three groups of Human renal tubular epithelial cells(HK-2)induced by high glucose ROS,MDA,SOD and nuclear factors-?B p65(NF-?B p65),I?K?,p-I?B?,Monocytechemotatic protein-1(MCP-1),Intercellular cell adhesion molecule-1(ICAM-1),E-cadherin,alphasmooth muscle actin(?-SMA),protein expression of Qingshen granule in each dose;set whether inhibition of HK-2 cells induced by high glucose oxidative stress mediated by activation of NF-?B signaling channel,reduce the HK-2 cells transdifferentiation(EMT),anti renal interstitial fibrosis.Methods: Selected 6 male Japanese albino rabbits,body weight 2.5±0.2kg/each,all purchases of the Animal Center of Anhui Medical University.The rabbits randomly divided into drug group and blank group with 3 rabbits in each.The concentration of clear renal granular aqueous solution was 1.0g/ml and animals in drug group were given clear renal granular aqueous solution by 5ml/kg gavage,1 times a day,10 consecutive days.Last administration,we collected blood by heart puncture with a syringe,stewing2 hours,then 3000 rpm centrifuged 10 min,got the supernate,filtrated by 0.22?m micropore filter,inactivated by 56?of water bath treatment for 30 min and stored at-20? for further detections.The blank group were given normal saline and collected blood as above.HK-2 cells were purchased from China Center for Type Culture Collection(Wu Han),we used the second and third generation cells.The same generation cells were randomly divided to 7 groups,including control group,mannitol group,high glucose group,Qingshen granule low dose group,Qingshen granule medium dose group,Qingshen granule high dose group,pyrrolidine dithio formic acid(pyrrolidine dithiocarbamate and PDTC)group.Cellular morphology was observed by inverted phase contrast microscope;MTT assay were used to evaluate cell growth;ROS,MDA,SOD content were determined by flow cytometry;Electrophoretic mobility shift assay determine DNA binding activity of NF-?B p65;Western blot was detected in cells of NF-?B p65,p-I?Ba,I?Ka,MCP-1 and ICAM-1 the protein expression;Immunocytochemical method was detected in cells NF-?B p65,E-cadherin and ?-SMA expression.Results: 1.MTT results showed that after 48 hours: compared with control group,cell viability of high glucose group,Qingshen granule low,medium,high dose group and PDTC group decreased significantly,and the difference is significant(P<0.01);compared with high glucose group,cell viability of Qingshen granule low,medium,high dose group and 5?mol/L PDTC group increased significantly,and the difference is significant(P<0.01);cell viability of Qingshen granule low dose group was significantly higher than Qingshen granule medium,high dose group(P<0.01),but there was no difference between Qingshen granule low dose group and 5?mol/L PDTC group(P>0.05);no difference was found between Qingshen granule medium and high dose group(P>0.05);cell viability of Qingshen granule medium and high dose group was lower than PDTC group;no difference was found between control group and mannitol group(P>0.05).On the basis of MTT results,we chose 48 h and 5?mol/L PDTC group for the rest part of the study.2.Comparison of ROS?MDA and SOD: compared with control group,high glucose group,Qingshen granule low,medium,high dose group and PDTC group ROS and MDA level increased,and there was difference in statistics(P<0.05);compared with high glucose group,Qingshen granule low,medium,high dose group and PDTC group ROS and MDA level decreased significantly,and the difference is significant(P<0.05);Qingshen granule low dose group ROS and MDA level was significantly lower than Qingshen granule medium,high dose group(P<0.05),but there was no difference between Qingshen granule low dose group and PDTC group(P>0.05);compared with control group,high glucose group,Qingshen granule low,medium,high dose group and PDTC group SOD level increased,and the difference is significant(P<0.01);compared with high glucose group,Qingshen granule low,medium,high dose group and PDTC group ROS and MDA level increased significantly,and the difference is significant(P<0.01);Qingshen granule low dose group SOD level was significantly higher than Qingshen granule medium,high dose group(P<0.05),but there was no difference between Qingshen granule low dose group and PDTC group(P>0.05).3.Electrophoretic mobility shift assay determine DNA binding activity of NF-?B p65(EMSA method): compared with control group,the NF-?B p65 DNA binding activity of high glucose group,Qingshen granule low,medium,high dose group and PDTC group increased,and the difference is significant(P<0.01);compared with high glucose group,the NF-?B p65 DNA binding activity of Qingshen granule low,medium,high dose group and PDTC group decreased significantly,and the difference is significant(P<0.01);the NF-?B p65 DNA binding activity of Qingshen granule medium dose group was significantly lower than Qingshen granule low,high dose group(P<0.05)and the NF-?B p65 DNA binding activity of PDTC group was lower than Qingshen granule medium dose group(P<0.05);the NF-?B p65 DNA binding activity of control group was lower than mannitol group(P<0.05).4.Comparison of expression of NF-?B p65,p-I?B?,I?K? protein in cells from all groups(Western blot method):? Comparison of expression of NF-?B p65 protein: compared with control group,NF-?B p65 protein content of the other groups increased significantly,the difference between control group and Qingshen granule medium,high dose group,high glucose group was significantly(P<0.01),but no difference between control group and Qingshen low medium dose group,PDTC group,mannitol group(P>0.05);compared with high glucose group,NF-?B p65 protein content of Qingshen low dose group and PDTC group decreased significantly(P<0.01),but no difference between Qingshen low dose group and PDTC group(P>0.05);NF-?B p65 protein content of Qingshen medium dose group was lower than high glucose group(P>0.05);no difference between Qingshen medium dose group and Qingshen high dose group.?Comparison of expression of p-I?B? protein: compared with control group,p-I?B?protein content of high glucose group,Qingshen granule low,medium,high dose group and PDTC group increased,and the difference is significant(P<0.01),but no difference between control group and Qingshen granule low dose group(P>0.05);compared with high glucose group,p-I?B? protein content of Qingshen granule low,medium,high dose group and PDTC group decreased significantly,and the difference is significant(P<0.01);p-I?B? protein content of Qingshen granule low dose group was significantly lower than Qingshen granule medium,high dose group(P<0.01),but no difference between Qingshen granule low dose group and PDTC group(P>0.05);no difference between Qingshen medium dose group and Qingshen high dose group(P>0.05);no difference between control group and mannitol group(P>0.05).? Comparison of expression of I?K? protein: compared with control group,I?K?protein content of high glucose group,Qingshen granule low,medium,high dose group and PDTC group increased,and the difference between control and PDTC group is significant(P<0.05),but no difference between control group and Qingshen granule low dose group(P>0.05),the difference between the other groups and control group is significant(P<0.01);I?K? protein content of Qingshen granule low dose group was significantly lower than Qingshen granule medium,high dose group(P<0.05),but no difference between Qingshen granule low dose group and PDTC group(P>0.05);no difference between Qingshen medium dose group and Qingshen high dose group(P>0.05).5.Comparison of expression of MCP-1 and ICAM-1 protein in cells from all groups(Western blot method):?Comparison of expression of MCP-1 protein: compared with control group,MCP-1protein content of high glucose group,Qingshen granule low,medium,high dose group and PDTC group increased,and the difference is significant(P<0.01),but no difference between control group and mannitol group(P>0.05);compared with high glucose group,MCP-1 protein content of Qingshen granule low,medium dose group and PDTC group decreased,and the difference is significant(P<0.01),but no difference between Qingshen granule high dose group and high glucose group(P>0.05);MCP-1 protein content of Qingshen granule low dose group was lower than Qingshen granule medium,high dose group but no difference between Qingshen granule low dose group and PDTC group(P>0.05).? Comparison of expression of ICAM-1 protein: compared with control group,ICAM-1 protein content of high glucose group,Qingshen granule low,medium,high dose group and PDTC group increased,and there was difference in statistics(P<0.05),but no difference between control group and mannitol group(P>0.05);compared with high glucose group,ICAM-1 protein content of Qingshen granule low,medium dose group and PDTC group decreased,and the difference is significant(P<0.05),but no difference between Qingshen granule high dose group and high glucose group(P>0.05);ICAM-1 protein content of Qingshen granule low dose group was lower than Qingshen granule medium,high dose group,PDTC group,and the difference is significant(P<0.05).6.Comparison of expression of NF-?B p65 in cells from all groups(Immunocytochemical method): compared with control group,the positive cells rate of high glucose group,Qingshen granule low,medium,high dose group and PDTC group increased,and the difference is significant(P<0.01);compared with high glucose group,the positive cells rate of Qingshen granule low,medium,high dose group and PDTC group decreased,and the difference is significant(P<0.01);the positive cells rate of Qingshen granule low dose group was lower than Qingshen granule medium,high dose group(P<0.01);the positive cells rate of Qingshen granule medium dose group was lower than Qingshen granule high dose group(P<0.01);the positive cells rate of PDTC group was lower than Qingshen granule low dose group(P<0.05);but no difference between control group and mannitol group(P>0.05).7.Comparison of expression of E-cadherin in cells from all groups(Immunocytochemical method): compared with control group,the positive cells rate of high glucose group,Qingshen granule low,medium,high dose group and PDTC group decreased,and the difference is significant(P<0.01);compared with high glucose group,the positive cells rate of Qingshen granule low,medium dose group and PDTC group increased,and the difference is significant(P<0.01);the positive cells rate of Qingshen granule low dose group was higher than Qingshen granule medium,high dose group(P<0.01);Qingshen granule medium dose group was higher than Qingshen granule high dose group(P<0.01);the positive cells rate of PDTC group was higher than Qingshen granule low dose group(P<0.01);but no difference between control group and mannitol group(P>0.05).8.Comparison of expression of ?-SMA in cells from all groups(Immunocytochemical method): compared with control group,the positive cells rate of high glucose group,Qingshen granule low,medium,high dose group and PDTC group increased,and the difference is significant(P<0.01);compared with high glucose group,the positive cells rate of Qingshen granule low,medium dose group and PDTC group decreased,and the difference is significant(P<0.01);the positive cells rate of Qingshen granule low dose group was lower than Qingshen granule medium,high dose group and PDTC group(P<0.01);the positive cells rate of Qingshen granule medium dose group was lower than Qingshen granule high dose group(P<0.01).Conclusion:1.High glucose can induce EMT and was not influenced by osmolarity;2.Serum containing Qingshen granules can inhibits HK-2 cells EMT induced by high glucose;3.NF-?B signaling pathway can be mediated by oxidative stress and participated in the progress of EMT and renal interstitial fibrosis;4.Serum containing Qingshen granules can inhibit NF-?B signaling pathway which was mediated by oxidative stress,and increase SOD level,reduce ROS and MDA level in HK-2 cells induced by high glucose,and reduce the expression of NF-?B p65,p-I?B?,I?K?,MCP-1,ICAM-1,?-SMA in HK-2 cells induced by high glucose,increase the content of E-cadherin,and improve renal tubular EMT,and to delay the progress of renal interstitial fibrosis.5.The low dose group was better than the medium dose group and the high dose group in inhibiting NF-?B signaling pathway which was mediated by oxidative stress induced by high glucose,improving renal tubular EMT,and delaying the progress of renal interstitial fibrosis. |
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