| ObjectiveChronic kidney disease?CKD?is one of the public health problems that need to be solved urgently at present.Prevention and treatment of renal interstitial fibrosis is the core strategy for the treatment of CKD.Traditional Chinese medicine?TCM?is effective in the treatment of CKD.Due to the multi-component and multi-target of TCM,the research on the effective substances and the mechanism of TCM has great limitations.At present,the research on the pharmacodynamic substances and its mechanism of TCM is based on the single target of molecular biology or pharmacology.These studies can not systematically demonstrate the efficacy and mechanism of TCM.In recent years,the“omics”technology provides a new and effective method for the study of the mechanism of action of multi-component drug substances in TCM.Lipomics is an important branch of metabonomics.Lipomics is mainly concerned with lipid metabolism in vivo.The changes of lipid metabolism were compared to identify biomarkers and reveal the mechanism of lipid metabolism.Lipidomics has an advantage in the study of multiple components of TCM through multi-organs,multi-systems,and multi-targets.It provides scientific technical support for the study of the mechanism of action of TCM,and also provides a new method for the development of TCM theory.In order to find the mechanism of Rhubarb?RH?in the treatment of CKD,this study was carried out based on lipomics.Many Chinese medicinal formulaes which are widely used for treatment of CKD contain RH.However,up to now,the mechanism of RH against renal fibrosis is rarely studied by combining“omics”and molecular biology.In this study,we will conduct molecular biology and lipidomics study to clarify the anti-renal injury effect of RH,Aloe-emodin?AE?,and Emodin?EM?on the animal,cellular and molecular levels.These studies provide a theoretical basis and experimental support for the clinical application of science,for the interpretation of TCM to provide new ideas and methods for the prevention and treatment of CKD.MethodsIn this study,we used UUO model of C57BL/6 mice and TGF-?1 model of HK-2cells.We first evaluated the pharmacodynamics of RH in vivo.Secondly,we evaluated the pharmacodynamics of AE and EM in vivo.Thirdly,we conducted the lipidomics studies,searching for differential biomarkers,networks and pathways in its main role.At last,we verified the pathways of major potent substances.The experimental contents are mainly divided into the following four parts:1.Pharmacodynamic evaluation of renal protective effect of RHIn vivo study:Male C57BL/6 mice?n=24?were selected and randomly divided into Sham group?n=8?,UUO group?n=8?,UUO+RH group?n=8?.The mice in each group were ingested the suspensions by gavage for 14 days after surgery.UUO+RH were given 3 mg·kg-1 after UUO operation.C57BL/6 mice were detected physiological and biochemical indicators.And C57BL/6 mice were observed the pathological morphology of renal injury by HE staining after administration.2.Renal protective effect of AE and EMIn vivo study:Male C57BL/6 mice were selected and randomly divided into Sham group?n=8?,UUO group?n=8?,UUO+LST group(10 mg·kg-1,n=8)and UUO+AE/EM group(20 mg·kg-1,n=8).The mice in each group were ingested the suspensions by gavage for 14 days after surgery.C57BL/6 mice were detected physiological and biochemical indicators.And C57BL/6 mice were observed the pathological morphology of renal injury by HE,PAS,and Masson staining after administration.Immunohistochemistry,western blot and RT-PCR technique were used to observe the changes of fibrosis related molecules TGF-?1,?-SMA,Collagen I,Collagen IV,E-cadherin and Fibronectin.The effect of AE and EM against renal fibrosis was evaluated.3.Lipomics study of AE against renal fibrosisMale C57BL/6 mice were selected and randomly divided into Sham group?n=8?,UUO group?n=8?,and UUO+AE group(20 mg·kg-1,n=8).The mice in each group were ingested the suspensions by gavage for 14 days after surgery.Lipidomics study of AE was based on UPLC-Q Exactive-MS technique.All of the data were introduced to the EZinfo 4.0 software for PCA,PLS-DA,and OPLS-DA to evaluate the AE treatment of renal injury.Through multivariate analysis results,combined with VIP values and t-test results,potential biomarkers were screened and compounds were identified by Lipidmaps,HMDB,and MetaboAnalyst 4.0 databases.KEGG database was used to analyze metabolic pathways of differential metabolites and to compare the signal pathways involved in differential metabolites.4.The renal protection mechanism of AE and EM.In vivo study:Male C57BL/6 mice were selected and randomly divided into Sham group?n=8?,UUO group?n=8?,UUO+LST(10 mg·kg-1,n=8)and UUO+AE/EM(20mg·kg-1,n=8)group.The mice in each group were ingested the suspensions by gavage for 14 days after surgery.The changes of autophagic and lipid droplets in renal tissue were observed by transmission electron microscopy.Western blot and RT-PCR were used to detect the autophagic marker molecule LC3,LC3 II/LC3 I,and Beclin-1.Western blot and RT-PCR were used to detect PI3K,p-Akt,and mTOR.In vitro study:The TGF-?1 model of HK-2 cells was used to simulate kidney injury process in vitro.The cells were divided into 3 groups:control group,TGF-?1group,and TGF-?1+AE/EM?40?M?group.Flow cytometry was used to observe the effect of AE on cell apoptosis.Western blot and RT-PCR were used to detect the autophagic marker molecule LC3,LC3 II/LC3 I,and Beclin-1.Western blot and RT-PCR were used to detect PI3K,p-Akt,and mTOR.Results1.Compared with UUO group,RH group could decrease serum creatinine from126.4±11.34?mol·L-1 to 72.03±7.208?mol·L-1?p<0.05?,and could reduce urea from 21.98±2.891?mol·L-1 to 16.91±1.538?mol·L-1?p<0.05?.RH can reduce the loss of renal epithelial cells,reduce interstitial inflammatory cell infiltration and reduce renal fibrosis.The results of studies show that RH has a certain renal protection,which can significantly improve physiological and biochemical indicators,reduce renal fibrosis.2.Compared with UUO group,AE and EM group could decrease serum creatinine from 119.4±12.76?mol·L-1,122.5±13.50?mol·L-11 to 83.38±5.012?mol·L-1,81.63±4.749?mol·L-1?p<0.05?,and could reduce urea from 20.74±3.691?mol·L-1,20.74±3.691?mol·L-1 to 15.15±0.977?mol·L-1,15.15±0.977?mol·L-1?p<0.05?,and could reduce renal tubule injury score from 11.6±1.00,11.7±1.00 to 9.44±0.88,9.67±0.71?p<0.05?,respectively.AE and EM can reduce the loss of renal epithelial cells,reduce interstitial inflammatory cell infiltration and reduce renal fibrosis.Western blot and RT-PCR results show that AE and EM could regulate the expression of renal fibrosis molecules.The results of studies show that AE and EM have renal protection,which can significantly improve physiological and biochemical indicators,reduce renal fibrosis,reduce renal tubular injury and renal fibrosis score,improve renal fibrosis molecule expression.The results of studies show that after TGF-?1 modeling,HK-2 cells grew slowly than that of the control group.Compared with the TGF-?1 group,AE could decrease percentage of apoptotic cells from 12.78%±0.84%to 8.88%±0.67%?p<0.05?.Western blot and RT-PCR results show that AE and EM could regulate the expression of renal fibrosis molecules?p<0.05?.The optimal drug concentration is 40?M by concentration screening.After AE and EM administration,the cells were significantly improved,the apoptosis was less,and renal fibrosis molecule expressionis were regulated.The results suggest that AE and EM have antirenal fibrosis effects.3.The protective mechanism of AE in UUO mice was based on lipidomics technology.The results of lipidomics study showed that AE could change the trend of endogenous lipid metabolites in mice.20 potential biomarkers were identified and screened from 660 positive and negative ions?p<0.05?.Network pathway analysis has revealed that these 20 biomarkers were mainly involved in regulation of autophagy,glycerophospholipid metabolism,metabolic pathways and so on.Autophagy pathway is a key pathway for AE to regulate lipid metabolism disorder through metabolic network map.4.The results of studies show that AE and EM can activate autophagy,increase autophagosomes and lipid droplets in renal tissue.Western blot and RT-PCR results show that AE and EM could up-regulate the expression of LC3,LC3 II/LC3 I,and Beclin-1?p<0.05?.AE and EM could down-regulate the expression of PI3K,p-Akt,and mTOR?p<0.05?.Western blot,and RT-PCR results show that AE and EM could up-regulate the expression of LC3,LC3 II/LC3 I,and Beclin-1 in HK-2 cells?p<0.05?.AE and EM could down-regulate the expression of PI3K,p-Akt,and mTOR in HK-2cells?p<0.05?.Conclusions1.In this study,the pharmacodynamic evaluation of RH,AE,and EM in vivo and in vitro were performed by pharmacodynamic evaluation.The results showed that RH,AE,and EM can significantly reduce the parameters of renal injury in mice and reduce renal fibrosis injury.AE and EM have a protective effect on the kidney.AE and EM have a strong kidney protection.They can promote the growth of HK-2 cells and reduce apoptosis.2.Based on UPLC-Q Exactive-MS technology,lipidomic analysis was performed after administration of AE.After analysis of the differential metabolite network pathway,the autophagy was the key pathway to resist fibrosis of AE.AE can inhibit PI3K/Akt/mTOR signaling pathway.The molecular biology experimental results are consistent with the results of lipidomic analysis.This study advances the knowledge of the overall efficacy and mechanism of RH and its components in the treatment of CKD.The results have expanded the therapeutic strategy of RH and its efficacy components in the prevention and treatment of CKD.Taken together,the study will provide theoretical and experimental basis for anti renal fibrosis that treated by components of TCM. |
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