Dapagliflozin restores autophagy and attenuates apoptosis via the AMPK/mTOR pathway in diabetic nephropathy rats and high glucose-induced HK-2 cells.

Diabetic nephropathy (DN) is a serious microvascular complication of diabetes mellitus. Significantly reduced levels of autophagy in diabetic kidneys play an important role in the development of DN. The present study investigated the effects of dapagliflozin (DAP) on renal autophagy and AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway in vivo and in vitro. We explored the effect of DAP in streptozotocin (STZ)-induced DN rats. The anti-DN effect of DAP was assessed by body weight, kidney weight/body weight ratio, blood and urine biochemical parameters, and pathological changes of kidney tissue. Number of autophagosomes in the kidney was investigated through Transmission electron microscopy. Besides, cell viability and apoptosis of DAP alone or combined with Compound C (CC, a selective AMPK inhibitor)-treated high glucose (HG)-induced HK-2 cells were detected by Cell Counting Kit-8 (CCK-8) and flow cytometry assays. Immunohistochemistry, Western blot, Enzyme-linked immunosorbent assay (ELISA), and immunofluorescence were employed to detect the expression levels of extracellular matrix (ECM) deposition, autophagy, apoptosis, and AMPK/mTOR pathway-associated targets in vivo and in vitro. The results showed that DAP ameliorated the body weight and decreased kidney weight, fasting blood glucose, and serum/urine biochemical parameters of renal damage, as well as renal pathological changes. Moreover, DAP significantly ameliorated HG-induced cell apoptosis and ECM deposition in HK-2 cells. However, these favorable effects of DAP could be abolished by co-treatment with CC in HG-induced HK-2 cells. Mechanistically, DAP can enhance autophagy in DN including increased LC3-II/I ratio, Beclin-1, p-AMPK protein levels, and decreased p62 and p-mTOR protein expressions, as well as inhibited renal fibrosis and apoptosis. In summary, DAP alleviated fibrosis, apoptosis, and autophagy in DN rats and HG-induced HK-2 cells by regulating the AMPK/mTOR pathway.

Ye YY ，Chen Y ，Yang J ，Wu J ，Wang P ... -  《-》

Piezo1 Modulates Neuronal Autophagy and Apoptosis in Cerebral Ischemia-Reperfusion Injury Through the AMPK-mTOR Signaling Pathway.

Cerebral ischemia-reperfusion (I/R) injury is a complex pathophysiological process involving multiple mechanisms, including apoptosis and autophagy, which can lead to significant neuronal damage. PIEZO1, a stretch-activated ion channel, has recently emerged as a potential regulator of cellular responses to ischemic conditions. However, its role in neuronal cell survival and death during ischemic events is not well elucidated. This study aimed to ascertain the regulatory function of PIEZO1 in neuronal cell apoptosis and autophagy in an in vitro model of hypoxia-reoxygenation and an in vivo model of brain I/R injury. HT22 hippocampal neuronal cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) to simulate ischemic conditions, with subsequent reoxygenation. In vitro, PIEZO1 expression was silenced using small interfering RNA (si-RNA) transfection. The effects on cell viability, apoptosis, and autophagy were assessed using CCK-8 assays, PI-Annexin/V staining combined with flow cytometry, and Western blot analysis. Additionally, intracellular Ca2+ levels in HT22 cells were measured using a Ca2+ probe. The involvement of the AMPK-mTOR pathway was investigated using rapamycin. For in vivo validation, middle cerebral artery occlusion/reperfusion (MCAO/R) in rats was employed. To determine the neuroprotective role of PIEZO1 silencing, sh-PIEZO1 adeno-associated virus was stereotaxically injected into the cerebral ventricle, and neurological and histological outcomes were assessed using neurological scoring, TTC staining, H&E staining, Nissl staining, and immunofluorescence. In HT22 cells, OGD/R injury notably upregulated PIEZO1 expression and intracellular Ca2+ levels. Silencing PIEZO1 significantly diminished OGD/R-induced Ca2+ influx, apoptosis, and autophagy, as indicated by lower levels of pro-apoptotic and autophagy-related proteins and improved cell viability. Additionally, PIEZO1 modulated the AMPK-mTOR signaling pathway, an effect that was counteracted by rapamycin treatment, implying its regulatory role. In vivo, PIEZO1 silencing ameliorated brain I/R injury in MCAO/R rats, demonstrated by improved neurological function scores and reduced neuronal apoptosis and autophagy. However, these neuroprotective effects were reversed through rapamycin treatment. Our findings indicate that PIEZO1 is upregulated following ischemic injury and facilitates Ca2+ influx, apoptosis, and autophagy via the AMPK-mTOR pathway. Silencing PIEZO1 confers neuroprotection against I/R injury both in vitro and in vivo, highlighting its potential as a therapeutic target for stroke management.

Yue Y ，Chen P ，Ren C 《-》

Fingolimod alleviates type 2 diabetes associated cognitive decline by regulating autophagy and neuronal apoptosis via AMPK/mTOR pathway.

This study aimed to reveal the role of fingolimod (FTY720) in mice with type 2 diabetes-associated cognitive decline and explore its potential neuroprotective mechanism. Mice were divided into five groups: normal control, normal control + FTY720 (1.0 mg/kg/day), type 2 diabetes mellitus, type 2 diabetes mellitus + low-dose FTY720 (0.5 mg/kg/day), and type 2 diabetes mellitus + high-dose FTY720 (1.0 mg/kg/day). Different doses of FTY720 were administered daily for 8 weeks after the induction of type 2 diabetes using a four-week high-fat diet feeding combined with continuous low-dose intraperitoneal injections of streptozotocin. After 8 weeks of treatment, the body weights and fasting blood glucose levels of mice from the five groups were compared. Morris water maze and new object recognition tests were used to evaluate cognitive function. Pathological changes in the hippocampal CA1 region were observed using haematoxylin-eosin and Nissl staining, and the ultrastructure of the hippocampal neurones was assessed using transmission electron microscopy. The expression levels of autophagy- and apoptosis-related proteins, such as LC3, Beclin-1, P62, Bax, and Bcl-2, in the mice hippocampus were detected by western blotting. Simultaneously, AMPK/mTOR signaling pathway proteins were detected to understand the potential mechanism. FTY720 had no significant effect on the body weight or fasting blood glucose levels in mice with type 2 diabetes. However, both FTY720 doses improved the cognitive function and hippocampal damage. In addition, the results suggested that FTY720 dramatically decreased P62 and Bax levels and increased LC3 II/LC3 I ratio, Beclin-1, and Bcl-2 expression in the hippocampus of type 2 diabetic mice. FTY720 also affected the expression of the AMPK/mTOR signaling pathway. Thus, FTY720 improved cognitive function and hippocampal pathological changes in type 2 diabetic mice without affecting fasting blood glucose levels. Our results show that FTY720 may exert neuroprotective effects in vivo by enhancing hippocampal autophagy and inhibiting apoptosis via the AMPK/mTOR signaling pathway.

Li J ，Yin M ，Wang Z ，Xiong Y ，Fang X ，Fang H ... -  《-》

Isoquercitrin improves diabetes nephropathy by inhibiting the sodium-glucose co-transporter-2 pathway.

Diabetic nephropathy (DN) is one of the most severe kidney complications and the primary contributor to end-stage renal disease on a global scale. It exacerbates the morbidity, mortality, and financial burden for individuals with diabetes. Isoquercitrin, a natural compound found in various plants, has demonstrated potential as an antidiabetic agent. However, it remains uncertain whether isoquercitrin exerts a protective effect on DN. Therefore, the objective of this study was to explore whether isoquercitrin confers a protective effect on DN and its potential mechanism. In vivo, a mouse model of DN induced by streptozotocin was established in the study. The hypoglycemic effect of isoquercitrin was assessed by measuring fasting blood glucose levels, insulin tolerance tests, and glucose tolerance test in animals. Urinary albumin creatinine ratio, serum lipid levels, and pathological changes in renal tissues were measured to evaluate the protective effect of isoquercitrin against DN. The expression of Sodium glucose co-transporter-2(SGLT2) was analyzed using real-time quantitative PCR and immunohistochemistry. The studies suggest that isoquercitrin significantly reduces fasting blood glucose levels, enhances the body's capacity to regulate blood glucose and insulin resistance, and facilitates renal pathology and renal function. Simultaneously, it can lower blood lipids (total cholesterol and triglyceride) and improve the risk factors of DN. Meanwhile, isoquercitrin suppressed the expression of SGLT2 in renal tissues of DN mouse models. In vitro, real-time quantitative PCR and Western blot were used to detect the expression of SGLT2 in the human renal tubular epithelial (HK-2) cells. The effects of isoquercitrin on the survival rate and glucose uptake capacity of HK-2 cells were determined by Cell-Counting-Kit-8 and glucose uptake methods. The results demonstrate that isoquercitrin suppressed the up-regulation of SGLT2 mRNA and protein in high-glucose-induced HK-2 cells. Additionally, isoquercitrin inhibited glucose uptake in HK-2 cells and mitigated high-sugar-induced damage. Thus, this study has concluded that isoquercitrin exhibits hypoglycemic and renal protective effects by inhibiting the SGLT2 pathway, indicating its potential as a promising anti-DN drug deserving further clinical investigation.

Zhang W ，Zhang Y ，Lv W ，Kong Z ，Wang F ，Wang Y ... -  《-》

Isoferulic acid regulates CXCL12/CXCR4-mediated apoptosis and autophagy in podocyte and mice with STZ-induced diabetic nephropathy.

Diabetic nephropathy (DN) is the most common microvascular complication of diabetes mellitus and a major cause of end-stage renal disease. Isoferulic acid (IFA) is a phenolic compound that has strong antioxidant, anti-inflammatory, and hypoglycemic effects. Researches and our previous study showed the potential anti-diabetic capacity and anti- oxidative stress damage targeting podocytes of IFA. The purpose of this study was to investigate whether IFA protects MPC5 podocytes from high glucose damage and alleviates DN symptoms in STZ-induced mice, as well as to explore the mechanism. The findings revealed that IFA (10, 25, 50 μM) significantly reduced high glucose-mediated toxicity, abnormal motility and morphology, ROS release, Ca2+ elevation with MPTP opening, apoptotic alterations with Caspase-3/7 activity increase and CXCL12 chemotaxis and interaction with CXCR4 in MPC5 podocytes. Furthermore, IFA increased Podocalyxin and LC3 II/I ratio. Meanwhile, IFA suppressed p53, mTOR, CASK, and p62. Furthermore, IFA has the ability to directly influence downstream mTOR, p53, and CASK apoptotic and podocyte motility regulatory targets when inhibiting the CXCL12/CXCR4 signaling pathway. In the sequent in vivo experiment, the results showed STZ-induced DN mice had higher kidney index, urination, UACR, lipid metabolism abnormalities and renal dysfunction, raised blood glucose, and podocyte damage than normal C57BL/6 mice. However, IFA treatment (50 mg/kg, 25 mg/kg, and 12.5 mg/kg) for 10 weeks restored the DN symptoms in the mice. IFA treatment elevated LC3B and LC3 II/I ratios and decreased p62 via suppressing chemokine axis CXCL12/CXCR4 with PI3K/Akt/mTOR, MMP9, and NF-κB p65 and activating podocyte markers WT1, nephrin, and Podocalyxin, thereby inducing autophagy and mitigating apoptosis in the DN mice kidneys. These findings suggest that IFA protective mechanism on kidney and podocytes simulating DN symptoms is primarily mediated by the CXCL12/CXCR4 pathways with the inactivation of apoptotic pathways and activation of autophagy.

Liu J ，Chang A ，Peng H ，Huang H ，Hu P ，Yao A ，Yin X ，Qu C ，Ni B ，Dong X ，Ni J ... -  《-》