Inotodiol Ameliorates Oxidative Stress and Apoptosis by Regulating PI3K/Akt/GSK-3β Signaling Pathways in Diabetic Nephropathy

Diabetic nephropathy (DN), a kind of microvascular complication, is a primary cause of end-stage kidney disease worldwide. However, therapeutic drugs for DN treatment are still in lack. Inotodiol (INO), a kind of lanostane triterpenoid isolated from INO that has various biological activities. In this study, we employed db/db mice as the spontaneous DN model in vivo, and high glucose treated MPC5 cells in vitro to elucidate the protective effects and underlying mechanisms of INO in DN. Ratio of right kidney weight/body weight was calculated, and levels of FBG, urine albumin/creatinine (UACR), BUN and Scr were measured. The SOD, CAT, GSH-Px and MDA levels in kidney were detected by using commercial kits. The histopathological changes of renal tissues were assessed by HE, PAS and Masson staining. The intracellular ROS was detected by using fluorescence probe DCHF-DA. Cytotoxicity assay was performed using CCK-8 assay kit. The rate of apoptosis was detected by flow cytometry. The expressions of Bax, Bcl-2, Cytc, Cleaved caspase-3, GSK-3β, pSer-GSK-3β, Akt, p-Akt, Synaptopodin, WT-1, Nrf2, NQO1, Keap1, heme HO-1 were measured by western blot. The expressions of Bax, CytC, WT-1, Synaptopodin, Bcl-2, GSK-3β and pSer9-GSK-3β in renal tissues were measured by immunohistochemistry. Our results showed that INO treatment reduced the FBG, BUN, Scr and UACR levels in db/db mice. Moreover, INO increased the expressions of Synaptopodin and WT-1 proteins. Besides, INO treatment also mitigated kidney histopathological changes, reduces kidney oxidative stress as reflected by reduced levels of Keap-1, NOX4 and MDA, but increased levels of kidney antioxidants SOD, CAT, GSH-Px, Nrf2, NQO1 and HO-1. Additionally, kidney apoptosis decreased as reflected by decreased protein levels of Cytc, Bax and Cleaved caspase-3 while its anti-apoptosis Bcl-2 protein levels increased. Mechanistically, INO inhibited GSK-3β activity by activating the PI3K/Akt signaling pathway, increased the level of anti-apoptosis, decreased level of oxidative stress and reduced podocyte injury in vivo and in vitro. Collectively, these results indicated that INO protected against DN through ameliorating oxidative stress and apoptosis via the PI3K/Akt/GSK-3β pathway.