Effect of Zishenwan on Pyroptosis and Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells in Diabetic Nephropathy Mice / 中国实验方剂学杂志

ABSTRACT

Objective:To study the efficacy and mechanism of Zishenwan （ZSW） against pyroptosis and epithelial-mesenchymal transition （EMT） of renal tubular epithelial cells in diabetic nephropathy （DN） mice， so as to provide evidence for the treatment of DN with ZSW. Method:The <italic>db/db</italic> mice with spontaneous diabetes were randomly divided into the model group， dapagliflozin （1.0 mg·kg^-1） group， and high-， medium-， and low-dose （6.0， 3.0， 1.5 g·kg^-1） ZSW groups. The non-diabetic <italic>db/m</italic> mice were classified into the normal group. The ones in the model and normal groups were given an equal volume of deionized water by gavage， while those in the other groups were intervened with the corresponding drugs for 12 weeks. The fasting blood glucose （FBG） level was tested at tail vein once every two weeks. The levels of urine albumin-creatinine ratio （ACR）， <italic>β</italic>-N-acetyl-D-glucosaminidase （NAG）， and cystatin C （CysC） were detected once every four weeks. After 12 weeks of administration， the blood sampled from eyeballs was used for measuring the blood urea nitrogen （BUN） and serum creatinine （SCr）. The pathological changes in renal tissues were observed by light microscopy and transmission electron microscopy. The expression of EMT markers in the renal tubular epithelium was analyzed by immunohistochemistry （IHC）. The in situ terminal end-labeling （TUNEL） staining was conducted to analyze the nuclear damage of renal tubular epithelial cells. The protein and mRNA expression levels of EMT markers， nucleotide-binding oligomerization domain-like receptor protein 3 （NLRP3） inflammasome and pyroptosis-related inflammatory cytokines in renal tissues were separately assayed by Western blot and Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）. Result:Compared with the normal group， the model group displayed significantly increased FBG， BUN， serum SCr， ACR， NAG， and CysC （<italic>P</italic><0.01）， impaired renal tissues， altered EMT marker expression intensities and levels （<italic>P</italic><0.01）， and elevated TUNEL-positive rate and protein and mRNA expression levels of pyroptosis-related inflammatory cytokines and NLRP3 inflammasome （<italic>P</italic><0.01）. Compared with the model group， ZSW and dapagliflozin significantly decreased the levels of FBG， BUN， serum SCr， ACR， NAG， and CysC （<italic>P</italic><0.01）， relieved the pathological injuries in renal tissues， changed the EMT marker expression intensities （<italic>P</italic><0.01） and protein and mRNA expression levels （<italic>P</italic><0.05， <italic>P</italic><0.01）， and down-regulated the TUNEL-positive rate （<italic>P</italic><0.01） of renal tubular epithelial cells as well as the protein and mRNA expression levels of pyroptosis-related inflammatory cytokines （<italic>P</italic><0.01） and NLRP3 inflammasome （<italic>P</italic><0.05， <italic>P</italic><0.01）. Conclusion:ZSW alleviates DN possibly by inhibiting pyroptosis and EMT in renal tubular epithelial cells.