| Objectives:1.In this study,the effect of "JHZP catabolism" disorder on glomerular mesangial cells and the intervention mechanism of Xieriga-4 were analyzed through cell experiments.2.The effective material basis of Xieriga-4 in protecting renal function was analyzed by Ussing chamber combined with HPLC-Q-TOP/MS technology.Methods:1.Forty-eight SD rats(SPF),male,weighing 180～220g,were randomly divided into 6 groups according to their weight after 5 days of adaptation to the environment,with 8 rats in each group.Which were normal group,model group,Xieriga-4(low,middle and high dose)groups and positive control group.The normal group and model group were given 0.5%CMC-Na solution,Xieriga-4(low,middle and high dose)groups were given corresponding dose of Xieriga-4 0.5%CMC-Na suspension,and the positive control group was given Shen Yan Xiao Zhong Pian tablets 0.5%CMC-Na suspension for 15 days and bathed at 40℃ for 1 hour every day.After the final dose,fasted for 24h with free access to water,each rat,except the normal group,was anaesthetized(Nembutal 30mg·kg-1)and the pylorus was ligated.After the operation,the rats were kept in separate cages,fasted and banned water for 17 h.Then all rats were anaesthetised,the kidney was fixed in 10%formaldehyde solution and prepared for pathological sectioning.Blood was taken from the abdominal aorta,centrifuged and the serum was stored at-80℃ for biochemical analysis.Histological observation of rat kidney tissue using HE staining,and the serum levels of URE and CRE in each group of rats were measured using an automatic biochemical instrument.2.Partial serum of rats in each group was inactivated in 56℃ water bath for 30min,and the cells were sterilized by microporous membrane(0.22μm).The cells were divided into 7 groups,namely normal control group,normal serum group,model serum group,Xieriga-4 lowdose medicated serum group,middle-dose medicated serum group,high-dose medicated serum group and positive drug-containing serum group.Except for the normal control group,the other cells were given the corresponding group serum,and the influence of each group serum on the cell survival rate was detected by CCK-8 method;The levels of URE and CRE in cell supernatant of each group were detected by automatic biochemical analyzer;Western blot was used to detect the expression of Slc22a5 and Mrps9 proteins in the cells.3.Twelve SD rats(SPF),male,weighing 180～220g,were randomly divided into 2 groups according to their weight after 5 days of adaptation to the environment,with 6 rats in each group.Namely normal group and model group.The model group was bathed at 40℃ for 1h per day for 15 days and then fasted for 24h with free access to water,each rat,except the normal group,was anaesthetized(Nembutal 30mg·kg-1)and the pylorus was ligated.After the operation,the rats were kept in separate cages,fasted and banned water for 17 h,then all rats were anaesthetised,fresh gastric and duodenal mucosa was taken and the transmembrane absorption Xieriga-4 samples were obtained by the Ussing Chamber experiment.Phenomenex Omega Polar C18 3.0*100 mm column was used with H2O(containing 0.05%FA)as the A mobile phase and methanol:acetonitrile(1:1)as the B mobile phase in a gradient elution at a flow rate of 0.3 ml-min-1 and an injection volume of 5 μl;the ESI ion source was used to collect data in positive and negative ion mode.MS primary mass number range of 80 to 1300m/z,MS/MS secondary debris collection pattern of 15 MS/MS 50 to 1300m/z,IDA informationdependent collection pattern;The use of real-time dynamic background deductions to obtain highly efficient secondary daughter ions;The ion source temperature was 550℃;The secondary collision energy was 40±20 eV.The chemical composition was identified based on primary high resolution mass spectrometry accurate mass-to-charge ratio data,secondary high resolution mass spectrometry fragment ions,the OS software secondary high resolution database and the open source MS Dial software.Results:1.The results of HE staining showed that the overall structure of renal tissue in normal group was basically normal,the glomerular structure was clear,no obvious degeneration such as atrophy and necrosis was found,no obvious degeneration such as edema,shedding and necrosis was found in renal tubular epithelial cells,and no obvious inflammatory cell infiltration was found;In the model group,the overall structure of renal tissue is abnormal,The tissue glomerulus is obviously atrophied and lobulated,more renal tubules are obviously dilated,and renal tubular epithelial cells are slightly edematous,prompt the success of modeling;Xieriga-4 low dose group,middle dose group,high dose group and positive control group all improved the pathological changes of renal tissue to varying degrees,and the overall structure of kidney tissue basically returned to normal.The results of the biochemical indexes showed that the levels of CRE and URE(p<0.01)were significantly higher in the model group compared with the normal group,and the levels of CRE and URE(p<0.01)were significantly lower in the low,medium and high dose groups of Xieriga-4 and positive control group.2.The results of the cell viability assay showed that the cell viability of the model serum group decreased significantly compared with that of the normal group(p<0.01);compared with the model serum group,the cell viability of HBZY-1 cells in each group with low,medium and high doses of Xieriga-4 containing serum intervention increased significantly(p<0.01),and the cell viability of the positive drug-containing serum group tended to increase,but it was not statistically significant.The results of biochemical indexes showed that compared with the normal group,the CRE and URE(p<0.01)levels in the model serum group were significantly increased;compared with the model serum group,the CRE(p<0.01)levels in the all doses of Xieriga-4-containing serum intervention group and the positive drug-containing serum group were significantly reduced,and the URE(p<0.05)levels in the low and high doses of Xieriga-4-containing serum intervention groups were significantly reduced.There was a tendency for the URE levels to decrease in the medium-dose serum-containing intervention group and the positive drugcontaining serum group,but it was not statistically significant.The results of protein blotting showed that the levels of Slc22a5(p<0.05)and Mrps9(p<0.01)were significantly lower in the model serum group compared with the normal group;compared with the model serum group,the levels of Slc22a5(p<0.01)were significantly higher in the all doses of Xieriga-4-containing serum intervention group and the positive drugcontaining serum group,and the levels of Mrps9(p<0.01)were significantly higher in the low dose of Xieriga-4 and the positive drug-containing serum group,and Mrps9(p<0.05)was significantly increased in the Xieriga-4 high-dose serum-containing intervention groups.3.HPLC-Q-TOP/MS results show that under the positive and negative ion mode,a total of 167 components of Xieriga-4 were identified,including 20 flavonoids and their glycosides,24 terpenoids and saponins,5 sugars,15 alkaloids,35 amino acids and other organic phenolic acid s,and 68 other components.There are 74 transmembrane absorbed components,including 5 fla vonoids and their glycosides,8 terpenoids and saponins,3 sugars,9 alkaloids,21 amino acids a nd other organic phenolic acids,and 28 other components.The top 10 components with high co ntent of Xieriga-4 are Berberine hydrochloride,Phellodendrine,Curcumin,Geniposide,Phellod endrine-1,Demethoxycurcumin,Bisdemethoxycurcumin,3-O-Feruloylquinic acid,Crocin I+Na,and Trehalose.The top 10 components with high transmembrane absorption content are,G en-iposide,Genipin,D-(+)-Glucose,Trehalose,Berberine hydrochloride,Phellodendrine,Phell odendrine-1,Betaine,Galactonic acid,Tauroursocholic acid,D-(-)quinic acid,[(2R)-3-hydrox y-2-oxoniopropyl]2-(trimethylazaniumyl)ethyl phosphate,Decylbenzene sulfonate,Xanthine,nicotinamide and 6-Hydroxy purine.To compare the content change trend of each component be fore and after normal intestinal mucosa,model intestinal mucosa,normal gastric mucosa and m odel gastric mucosa in rats,It is preliminarily judged that the main effective components of Xie riga-4 for protecting kidney are as follows:Berberine hydrochloride,Phellodendrine,Phelloden drine-1,Geniposide,D-(-)Quinic acid,Genipin and Trehalose.Conclusions:Firstly,pyloric ligation was effective in replicating kidney injury in rats and that the mechanism of injury may be linked to MRPS9 protein.Secondly,Xieriga-4 can effectively interfere with pyloric ligation-induced renal injury,and the effect of low-dose Xieriga-4 is the most significant.Finally,it is preliminarily speculated that Berberine hydrochloride,Phellodendrine,Phellodendrine-1,Geniposide,D-(-)Quinic acid,Genipin and Trehalose,which are the blood components of Xieriga-4,are the main active components for protecting kidney,and the transport of the above effective substances may be related to the protein transport function of SLC22A5. |
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