EA attenuates inflammatory response apoptosis in HG-stimulated RTECs and podocytes.
To determine whether EA protects against HG-induced RTEC and podocyte dysfunction, the cytotoxicity of EA was investigated in RTECs and podocytes exposure to HG (30 mM) for 24 h. As shown in Figure 1A, EA had no obvious inhibitory effect on cell viability when the concentration of EA less than 30 μM. Compared with NG (5 mM) group, cell viability was significantly inhibited by approximately 40% in the HG group. However, HG-induced growth inhibition of RTECs and podocytes was rescued by EA treatment (Figure 1A). Inflammatory cytokines, including TNF-α, IL-1β and IL-6, levels were significantly increased by HG stimulation; however, EA treatment sharply down-regulated HG-evoked inflammatory response in RTECs and podocytes (Figure 1B and 1C). We also found that EA treatment reduced the proportion of apoptotic cells in HG-stimulated RTECs and podocytes (Figure 1D).
EA mediates the NF-κB/miR-150-3p axis in HG-induced RTEC and podocyte apoptosis.
EA has been highlighted as a NF-κB inhibitor to alleviate multiple pathological processes, including DN . Moreover, NF-κB can modulate miR-150-3p expression in several diseases [25, 26]. Herein, we hypothesized that EA-mediated NF-κB/miR-150-3p might be implicated in HG-induced RTEC and podocyte apoptosis. As shown in Figure 2A, 2B and 2C, both nuclear NF-κB protein and miR-150-3p expression levels were significantly elevated in HG-stimulated RTECs and podocytes compared with those cells treated with NG. However, EA treatment inhibited HG-activated NF-κB and miR-150-3p in RTECs and podocytes. To further investigate the roles of miR-150-3p in HG-induced RTEC and podocyte apoptosis, miR-150-3p inhibitors were transfected into RTECs and podocytes to silence the expression of miR-150-3p. As shown in Figure 2D, transfection of miR-150-3p inhibitors significantly declined miR-150-3p expression in RTECs and podocytes compared with the control group. After transfection with miR-150-3p inhibitors into HG-stimulated RTECs and podocytes, our findings suggested that HG-induced apoptosis (Figure 2E) and growth inhibition (Figure 2F) were rescued by the inhibition of miR-150-3p expression.
BCL2 expression can be regulated by miR-150-3p and EA
In our study, based on online bioinformatics analysis and luciferase assays, our findings suggested that BCL2 is a direct target of miR-150-3p, reflecting that transfection with miR-150-3p mimics into BCL2 3’-UTR-WT podocytes and RTECs significantly reduced luciferase activity, but the luciferase activity had no obvious change in BCL2 3’-UTR-Mut cells (Figure 3A). In addition, overexpression or silencing of miR-150-3p significantly repressed or up-regulated the protein expression of BCL2 in podocytes and RTECs, respectively (Figure 3B). In vitro experimental measurements revealed that HG led to a significant reduction of BCL2 protein expression in podocytes and RTECs, while EA treatment reversed HG-induced BCL2 inhibition (Figure 3C and 3D). These findings indicate that EA-evoked BCL2 activation may be associated with the inhibition of the NF-κB/miR-150-3p axis in vitro.
EA has no effect on body weight and FBG in diabetic mice.
To further investigate the role of EA on renoprotective action, we performed a pharmacological experiment in diabetic mice with or without EA administration. Physiological parameters, body weight and FBG, exhibited that a significant decrease in body weight and an increase in FBG were observed in the experimental period after STZ injection (Figure 4A and 4B). However, EA administration for 8 weeks had no obvious effect on body weight and FBG in diabetic mice (Figure 4A and 4B).
EA mitigates renal injury in diabetic mice.
To explore the renal protective activity of EA on hyperglycemia-induced renal dysfunction, STZ mice were administrated with or without EA for 8 weeks. BUN and serum Cr (Figure 5A), mRNA expression of KIM1 and NGAL (Figure 5B), urinary KIM1 and NGAL (Figure 5C), and urinary total protein (Figure 5D) were significantly elevated in diabetic mice compared with those of normal mice. Podocyte injury marker nephrin mRNA expression was sharply reduced in the kidney of diabetic mice (Figure 5E). Intriguingly, administration of diabetic mice with EA for 8 weeks markedly improved these pathological parameters. Histologic examination by PAS staining and H&E staining exhibited that tubular atrophy (Figure 5F) and glomerular pyknosis (Figure 5G) were presented in diabetic mice. Histologic scoring indicated that tubular and glomerular injury were significantly attenuated in diabetic mice with EA treatment (Figure 5F and 5G).
EA alleviates interstitial fibrosis in diabetic mice.
As shown in Figure 6A, there was strong collagen deposition with blue staining in STZ-treated mice. However, the density of blue staining was obviously reduced in diabetic mice with EA administration (Figure 6A). Semiquantitative scoring validated that hyperglycemia-induced interstitial fibrosis in diabetic mice was dramatically mitigated by EA administration (Figure 6B). As shown in Figure 6C, 6D and 6E, up-regulation of fibrosis markers FN1, Col1a1 and Col3a1 mRNA expression levels in diabetic mice were significantly reversed by EA administration.
EA mediates NF-κB/miR-150-3p/BCL2 activity in diabetic mice.
EA-mediated the activation of BCL2 via inhibiting NF-κB/miR-150-3p axis has been observed in HG-stimulated podocytes and RTECs. Next, we further explore the effect of EA on NF-κB/miR-150-3p/BCL2 activity in the kidney of STZ-treated mice. We also found that hyperglycemia facilitated nuclear NF-κB protein and miR-150-3p expression and inhibited BCL2 protein expression in the kidney. However, hyperglycemia-induced up-regulation of NF-κB protein (Figure 7A) and miR-150-3p (Figure 7B) and down-regulation of BCL2 (Figure 7C) were reversed by EA treatment. In addition, hyperglycemia-induced up-regulation of pro-inflammatory cytokines and mediators, IL-1β (Figure 7D), IL-6 (Figure 7E) and MCP1 (Figure 7F), was strikingly reduced by EA administration.
The effect of miR-150-3p antagomir in kidney, heart and liver of diabetic mice.
To further investigate the role of miR-150-3p in the pathogenesis of DN, diabetic mice were treated by the injection of miR-150-3p antagomir. Compared with diabetic mice, miR-150-3p expression in the kidney, heart and liver was significantly reduced after received with anti-miR-150-3p treatment (Figure 8A). Histologic examination exhibited that anti-miR-150-3p administration markedly improved hyperglycemia-induced tubular and glomerular injury (Figure 8B). However, both STZ and anti-miR-150-3p treatment had no obvious detrimental effect on the morphology of heart and liver (Figure 8B) and serum ALT and AST (Figure 8C), suggesting that anti-miR-150-3p treatment had no obvious influence on cardiac and hepatic functions. Collectively, anti-miR-150-3p treatment ensures medication safety in vivo experiments.
Anti-miR-150-3p treatment mitigates renal injury in diabetic mice.
To explore the function of miR-150-3p antagomir, diabetic mice were received miR-150-3p antagomir treatment by tail intravenous injection biweekly for 8 weeks. As shown in Figure 9A, 9B and 9C, hyperglycemia-induced the up-regulation of renal injury marker, BUN, serum Cr and 24h urinary total protein, was decreased by anti-miR-150-3p injection. Histologic examination revealed that hyperglycemia-induced tubular injury (Figure 9D) and interstitial fibrosis (Figure 9E) were dramatically alleviated by anti-miR-150-3p injection. As shown in Figure 9F, fibrotic biomarker FN1 protein expression was significantly reduced in the kidney of diabetic mice after anti-miR-150-3p injection. Both nephrin (Figure 9G) and BCL2 (Figure 9H) protein expression was significantly increased in the kidney of diabetic mice after anti-miR-150-3p injection. Based on in vitro and in vivo findings, our study deducted that EA performs a renal protective activity, at least partly, by mediating the NF-κB/miR-150-3p/BCL2 signaling axis (Figure 10).