Effective Components and Mechanism of Cistanches Herba Aqueous Extract Against Diabetic Nephropathy Based on the Spectral-effect Relationship
OBJECTIVE To screen out the pharmacodynamic substances of Cistanches Herba aqueous extracts,explore the basis of the pharmacodynamic substances and their mechanism of action in the treatment of diabetic nephropathy(DN),based on the spectral relationship between the mass spectral peak areas of different elution sites of Cistanches Herba aqueous extracts and their anti-DN effects.METHODS UPLC-Orbitrap-MS/MS technique was used to characterise the chromatographic peaks;MTT method was used to detect the effects of different elution sites of Cistanches Herba aqueous extract on the proliferation of high glucose and high fat HK-2 cells;grey correlation analysis and partial least squares method were used to analyse the spectral relationship between mass spectrometry peak area and anti-DN activity.MTT method was used to determine the anti-DN activities of the individual components of Cistanches Herba aqueous extract;biochemical kit and ELISA were used to determine the levels of oxidative indicators(SOD,GSH-P)and inflammatory factors(IL-1β,TNF-a,TGF-p);Western blotting was used to detect the expression of apoptosis-related proteins.RESULTS UPLC-Orbitrap-MS/MS technique speculated and identified 72 common compounds;In Cistanches Herba aqueous extracts,water,20％ethanol,and 40％ethanol-eluted sites differentially increased the proliferation rate of HK-2 cells in a high-sugar,high-fat environment;Partial least squares and grey correlation analyses showed that the constituents of the aqueous extracts of Cistanches Herba with greater anti-DN contributions were 8-epideoxymatricinic acid,geniposidic acid,pinoresinol,betaine and syringin,et al.MTT assay reveals that 8-epi-deoxystrychnic acid and geniposidic acid had significant proliferative effects on HK-2 cells in a high glucose and high fat environment;Biochemical kit and ELISA showed that 8-epideoxystrychnic acid and geniposidic acid were able to up-regulate the activity of SOD and GSH-Px,and at the same time they had an inhibitory effect on the expression of inflammatory factors IL-1β,TNF-α and TGF-β.Western blotting results showed that 8-epideoxystrychnic acid and geniposidic acid were able to down-regulate and up-regulate the markers of dermal mesenchymal transition:α-SMA,Collagen Ⅰ and E-cadherin,and they could exert anti-DN effects by inhibiting the PI3K-Akt pathway.CONCLUSION The two compounds,8-epideoxystrychnic acid and geniposidic acid,which are screened by the spectroeffective relationship of anti-DN among the many chemical constituents contained in Cistanches Herba,can affect oxidative stress,inflammation and epithelial-mesenchymal transformation of renal tubular cells by inhibiting the PI3K-Akt signalling pathway,and improve renal pathology,degree of fibrosis and renal function,which will be useful for the in-depth study of aqueous extracts of Cistanches Herba in the treatment of DN.
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