Mechanism of Qianyang Yuyin Granules Regulating NR3C2/ROS/ERK Pathway to Alleviate Aldosterone-induced Podocyte Injury / 中国实验方剂学杂志

ObjectiveTo investigate the protective mechanism of Qianyang Yuyin granules （QYYY） on aldosterone-induced podocyte injury. MethodA total of 30 C57BL/6J mice were randomly divided into five groups： control group， model group， QYYY low dose （QYYY-L） group， QYYY high dose （QYYY-H） group， and spironolactone （SPL） group， with six mice in each group. Except for the control group， mice were implanted with osmotic minipumps and injected continuously with aldosterone （300 μg·kg-1·d-1） to induce renal injury. The drug administration group was given low and high doses （2.6， 5.2 g·kg-1·d-1） of QYYY and SPL （18 mg·kg-1·d-1） for 28 days. The renal pathological changes of mice were observed by hematoxylin-eosin （HE） staining and Masson staining. The expression levels of Nephrin， Desmin， B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X （Bax）， cleaved Caspase-3， nuclear receptor subfamily 3 group C member 2 （NR3C2）， extracellular regulated protein kinases （ERK）， and phospho-ERK （p-ERK） in kidney tissue were detected by Western blot. The apoptosis levels of kidney tissue were detected by TdT-mediated dUTP nick and labeling （TUNEL） staining， and the superoxide dismutase （SOD） levels were detected. In vitro， the mice were divided into five groups： Control group， model group （aldosterone concentration of 200 nmol·L-1）， QYYY-L group， QYYY medium dose （QYYY-M） group， and QYYY-H group （25， 50， and 100 mg·L-1）. The effect of different concentrations of QYYY on the relative viability of aldosterone-induced podocytes was detected by cell proliferation and viability assay （CCK-8）. The expressions of Nephrin， Desmin， Bax， Bcl-2， cleaved Caspase-3， NR3C2， and p-ERK/ERK were detected by Western blot. AnnexinV-FITC/PI flow cytometry was used to detect the apoptosis levels of podocytes. Reactive oxygen species （ROS） in podocytes were observed by DCFH-DA. ResultCompared with the control group， the model group showed structural pathological changes and fibrotic conditions in the kidney， increased apoptosis levels （P<0.01）， and decreased SOD levels （P<0.01）. Aldosterone concentration at 200 nmol·L-1 showed a significant decrease in podocyte activity （P<0.05）. Podocytes in the model group showed structural pathological changes， disordered arrangement of intercellular microfilaments， increased apoptosis levels （P<0.01）， and increased intracellular ROS levels （P<0.01）. The protein expressions of Nephrin， Bcl-2， and p-ERK/ERK in kidney tissue and podocytes were decreased （P<0.05， P<0.01）. The protein expressions of Desmin， Bax， cleaved Caspase-3， and NR3C2 were increased （P<0.05， P<0.01）. Compared with the model group， QYYY alleviated the structural damage and fibrosis of the kidney， decreased the apoptosis levels （P<0.05， P<0.01）， and enhanced the SOD content of the kidney （P<0.05， P<0.01）. QYYY improved the activity of podocytes （P<0.05， P<0.01）， restored the foot process structure of podocytes， and decreased apoptosis levels （P<0.01） and ROS levels of podocytes （P<0.01）. The protein expressions of Nephrin， Bcl-2， and p-ERK/ERK in kidney tissue and podocytes were increased （P<0.05， P<0.01）， and the protein expressions of Desmin， Bax， cleaved Caspase-3， and NR3C2 were down-regulated （P<0.05， P<0.01）. ConclusionQYYY improves aldosterone-induced podocyte injury by regulating the NR3C2/ROS/ERK pathway.

Investigation of Pharmacodynamic Material Basis and Mechanism of Qianyang Yuyin Granules Against Hypertensive Renal Damage Based on LC/Q-TOF-MS and Network Pharmacology / 中国实验方剂学杂志

Objective:To systematically study the chemical components of Qianyang Yuyin granules and explore its main pharmacodynamic substances and mechanism in the prevention and treatment of hypertensive renal damage. Method:Liquid chromatography coupled with quadrupole time-of-flight mass spectrometry （LC/Q-TOF-MS） was employed to comprehensively analyze the chemical components of Qianyang Yuyin granules. Agilent Poroshell 120 SB-C₁₈ column （3.0 mm×100 mm， 2.7 μm） was used， flow rate was 0.4 mL·min^-1， electrospray ionization （ESI） was applied and operated in positive and negative ion modes， the acquisition range was <italic>m</italic>/<italic>z</italic> 25-1 000. Mobile phase in positive ion mode consisted of water+10 mmol·L^-1 ammonium formate+0.125% formic acid+0.1% methanol （A）-［acetonitrile-water （9∶1）+10 mmol·L^-1 ammonium formate+0.125% formic acid］ （B）， and in negative ion mode consisted of water+10 mmol·L^-1 ammonium formate+0.1% methanol （A）-［acetonitrile-water （9∶1）+10 mmol·L^-1 ammonium formate］ （B） with the gradient elution （0-3.5 min， 5%B； 3.5-4 min， 5%-10%B； 4-9 min， 10%-25%B； 9-18 min， 25%-30%B； 18-25 min， 30%-50%B； 25-27 min， 50%-90%B； 27-32 min， 90%B； 32-33 min， 90%-5%B； 33-39 min， 5%B）. According to the information of the accurate mass， the multistage fragment ions， the mass spectrometric data of the standard substances and the relative reference literature， the structures of the chemical components in Qianyang Yuyin granules were identified. Based on the identified components， network pharmacology study， including target prediction and functional enrichment was applied to screen out the main active substances against hypertensive renal damage， and explore the potential mechanism. Result:A total of 99 chemical components were identified， from which 43 active substances and 48 key targets were screened out. The key components contained kaempferol， quercetin， ferulic acid， luteolin， caffeic acid methyl ester， cinnamic acid， aloe-emodin， emodin， gallic acid， <italic>N</italic>-<italic>trans</italic>-feruloyltyramine， isoorientin， 8-<italic>O</italic>-feruloylharpagide， ethyl caffeate， isookanin， cyasterone， 2，3，5，4'-tetrahydroxystilbene-2-<italic>O</italic>-<italic>β</italic>-<italic>D</italic>-glucopyranoside， loganin， alisol B-23-acetate and harpagide. The key targets included vascular endothelial growth factor A （VEGFA）， serine/threonine protein kinase 1 （AKT1）， Jun proto-oncogene （JUN）， etc. Conclusion:Qianyang Yuyin granules mainly exert the effects of removing heat from the liver， tonifying the kidney and removing blood stasis via modulation of vascular endothelium， angiogenesis， inflammatory reaction， oxidative stress， immune response and so on.