Study On The Effect Of Qijishenkang On The Mechanism Of Renal Tubular Epithelial Cells Induced By TGF-?₁

Purpose:Qi ji shenkang granule is derived from professor zhang jun's more than 30 years of clinical experience in the treatment of children's kidney disease in combination with the ancient formula.The objective of this study was to induce mesenchymal transdifferentiation of renal tubular epithelial cells by transforming growth factor factor 1,and then to prepare different groups of pharmacological serums for use on mesenchymal transdifferentiation of renal tubular epithelial cells.Three different time points were selected,namely,24h,48h and 72h.By observing the expression levels of FN,ColIV,e-cadherin and ?-sma proteins in renal tubular epithelial cells induced by TGF-?1,the effects of Qi ji shenkang on the mechanism of action of TGF-?1 induced renal tubular epithelial cells were investigated from the cellular and protein levels.Material and method:1 Material1.1 Experimental animals 54 SPF grade SD rats,half male and half female(liaoning changsheng biotechnology co.,LTD.).1.2 Cell line hk-2 human tubular epithelial cell line(guangzhou genio biotechnology co.,LTD.).1.3 Experimental drugs1.3.1 Qi ji shenkang group:xiaoji,white snake tongue grass,crane grass,peony skin,salvia miltiorrhiza,astragalus astragalus,haidongteng single taste medicine granules(jiangyin tianjiang pharmaceutical co.,LTD.).1.3.2 Control group:telmisartan tablets(Shanghai boehringer ingelheim pharmaceutical co.,LTD.).1.4 Main instruments and reagents1.4.1 Main instrumentsCo2 culture(Thermo USA);Ultra clean workbench(Thermo USA);Table centrifuge(Heraeus Germany);Enzyme marker(TECAN Switzerland);Inverted phase contrast microscope(Olympus Japan);Ultra-low temperature refrigerator(SANYO Japan);Swimming apparatus(liu yi instrument factory Beijing);Horizontal shaker(liu yi instrument factory Beijing);Vertical electrophoresis tank(liu yi instrument factory Beijing).1.4.2 Main reagentsRpmi-1640 medium(HyClone company);PBS:phosphate buffer(HyClone);Converted growth factor protecl(peprotech inc.,USA);Calf serum(HyClone company);ELISA kit(AMEKO,inc.);Rabbit anti-signaling-SMA polyclonal antibody(Cell Signaling inc.);Rabbit anti-e-cadherin polyclonal antibody(Cell Signaling,inc.);HRP labeled anti-rabbit IgG(Cell Signaling inc.).2 Methods2.1 Cell culture and passageCell culture:the growth state of human renal tubular epithelial cells was first observed,and then the original culture medium in the culture bottle was absorbed and the new complete culture medium was added for culture and breeding.The culture medium in the bottle was replaced every two days.Cell passage:when the cells cover about 80%of the bottom of the bottle and the cell growth cycle is logarithmic,the original culture medium in the bottle is sucked up,washed with PBS twice,then digested with 0.25%trypsin,and placed in a C02 incubator at 37? for 4-5min.After the digestion,the upper culture medium was absorbed,and the cells were shed in the culture medium by blowing the wall of the bottle repeatedly.Finally,all the culture liquid in the bottle was sucked out,the solution was discarded after centrifugation,and a new complete culture liquid was added to make cell suspension,which was equally added to a number of new culture bottles to complete cell passage.2.2 Preparation of pharmacological serumA total of 54 SD rats were randomly divided into 6 groups,with 9 rats in each group.The groups were the blank group,the model group,the telmisartan group,and the low,medium and high dose groups of qi ji shenkang granules.Gavage was performed for 5 consecutive days.Blood was collected from the abdominal aorta 1 hour after the last administration.Please centrifuge the blood.The specific administration methods were as follows:the blank group and the model group were given 0.9%normal saline by gavage.Telmisartan group was equivalent to adult maximum 80mg/d.The effective dose of qiji shenkang group was equivalent to that of human beings.The effective dose of qi ji shenkang in the concentration group was 2 times of that of human.Qi ji shenkang high concentration group equivalent to 4 times the effective dose.2.3 detection of cell activity(MTT method)According to the preliminary experimental results,the optimal concentration of drug-containing serum was 15%,and the optimal concentration of TGF-?1 was 10ng/mL Cell suspension was inoculated on a 96-well culture plate and divided into six groups,namely the blank group,the model group,the telmisartan group,and the qi ji shenkang low,medium and high dose groups,n=10.OD value(565mm)was measured at three time points of 24h,48h and 72h.2.4 Detection of FN and ColIV(ELISA method)Cell supernatant was collected and labeled at three time points of 24h,48h and 72h,respectively.FN and ColIV were detected by ELISA.In strict accordance with the operation instructions,24h and 72h n=7,48h n=8,grouping with MTT.Then measure the OD value at three time points and draw the standard curve according to the standard parameters.2.5 Detection of-sma and e-cadherin(Western Blot)Cells were collected at three time points of 24h,48h and 72h,and protein concentrations in each group were determined by coomassie bright blue method.Each sample is made into superscript solution.Pressure electrophoresis,membrane transfer,sealing,adding primary antibody,secondary antibody,film washing,luminescence and color rendering,image acquisition and preservation by Western Blot imaging system.Finally,Image J software is used to analyze the gray value.Results:1.Results of cell activity assay(MTT method)Compared with other 5 groups,the 24h high-dose group had statistical significance.The high dose group of 48h had statistical significance compared with the model group and the dose group of qi ji shenkang.Compared with telmisartan group,the 72h high dose group of qilyishenkang had statistical significance.Compared with the model group,the 24h and 48h high dose of qi ji shenkang had statistical significance.Compared with telmisartan group,the high-dose group of qi ji shenkang at three time points had statistical significance2 FN,ColIV test results(ELISA)2.1 FN content(ELISA method)At the three time points of 24h,48h and 72h,the model group had statistical significance compared with the normal group,the model group and the four drug groups.At 48h and 72h,telmisartan group had statistical significance when compared with the groups with different doses of qjishenkang,and at 24h telmisartan group had statistical significance when compared with the groups with medium and high doses of qjishenkang.The 48h and 72h qjishenkang low,middle and high school groups all had statistical significance.The low dose group of qi ji shenkang at three time points had statistical significance compared with the medium and high dose group of qi ji shenkang2.2 ColIV content(ELISA)At the three time points of 24h,48h and 72h,the model group had statistical significance compared with the normal group,the model group and the four drug groups.The 24h telmisartan group had statistical significance compared with the low dose group of qiliqishenkang,and the 48h and 72h telmisartan group had statistical significance compared with the different dose group of qiliqishenkang.The dosage of qjishenkang at 48h and 72h showed statistical significance.The low dose group of qi ji shenkang at three times had statistical significance compared with the medium and high dose group of qi ji shenkangTest results of e-cadherin and-sma(Western Blot)3.1 western blot analysis results of e-cadherinCompared with the model group,the remaining 5 groups had statistical significance Compared with telmisartan group,the low dose group of qi ji shenkang had statistical significance.Compared with the low dose group,the medium dose group and the high dose group had statistical significance3.2 Western Blot analysis results of-smaCompared with the model group,the remaining 5 groups had statistical significance Compared with telmisartan group,the low-dose and high-dose groups of qjishenkang had statistical significance.Compared with the low dose group,the expression of m-sma in the medium dose group and the high dose group was statistically significant.Compared with the medium dose group,the high dose group had statistical significanceConclusion:1.1.This experiment proved that TGF-?1 can induce the transdifferentiation of renal tubular epithelial cells into renal tubular epithelial-mesenchymal cells,and the transdifferentiation of renal tubular epithelial cells into mesenchymal cells can increase the production of extracellular matrix,thus promoting renal fibrosis2.This experiment proved that qi ji shenkang granules could effectively inhibit the occurrence of renal tubular epithelial-mesenchymal cell transdifferentiation and reduce the production of extracellular matrix,and its inhibitory effect was strengthened with the increase of dose3.This experiment proved that telmisartan can also effectively inhibit the occurrence of renal tubular epithelial-mesenchymal cell transdifferentiation and reduce the production of extracellular matrix.