Effects of maslinic acid on the characteristics of diabetic mice.
To demonstrate the potential therapeutic effects of maslinic acid on diabetes, body weight, fasting blood glucose levels, average food intake and average water intake were measured and analyzed. As shown in Fig. 1a, 20 mg/kg maslinic acid treatment significantly rescued the body-weight loss induced by STZ administration. Similarly, 5 mg/kg, 10 mg/kg and 20 mg/kg maslinic acid treatment markedly decreased the fasting blood glucose levels compared to the STZ group (Fig. 1b). In addition, 20 mg/kg maslinic acid significantly lowered the food and water intake in diabetic mice (Fig. 1c and d). Totally, administration of different amounts of maslinic acid alleviated the symptoms of diabetic mice to varying degrees, and there were no significant side-effects of maslinic acid on diabetic mice. Accordingly, we chose 20 mg/kg as the dosage for long-term maslinic acid administration.
Effects of maslinic acid on serum parameters in diabetic mice.
To investigate the effects of maslinic acid administration on the renal function, kidney/body weight, Urea, BUN and Creatinine in serum of mice with diabetic nephropathy was measured by an automatic analyzer (N = 14 per group). As shown in Fig. 2a, STZ treatment induced the renal fibrosis and the increase of kidney/body weight ratio. On the other hand, 20 mg/kg maslinic acid treatment significantly rescued the increased kidney/body weight ratio, suggesting that maslinic acid protected the nephron of mice with diabetic nephropathy from sclerosis. Similarly, 5 mg/kg, 10 mg/kg and 20 mg/kg maslinic acid treatment induced the decrease of serum urea, BUN and creatinine levels to varying degrees (Fig. 2b, c and d), suggesting that maslinic acid administration protect the renal function of mice with diabetic nephropathy.
Effects of maslinic acid on urine parameters in diabetic mice.
To further demonstrate the nephroprotective role of maslinic acid in diabetic nephropathy, urine parameters including urine volume, urine albumin, total proteins and creatinine of diabetic mice after 8-week maslinic acid treatment were measured and analyzed respectively. As shown in Fig. 3a, maslinic acid treatment alleviated the polyuria in diabetic mice induced by STZ. More importantly, maslinic acid treatment significantly lower albumin and total protein in the urine of diabetic mice, suggesting that maslinic acid protected the function of glomerulus in diabetic nephropathy (Fig. 3b and c). In addition, maslinic acid treatment also induced the increase of creatinine in urine of mice with diabetic nephropathy (Fig. 3d), indicating that maslinic acid ameliorated the filtration function of the kidney.
Effects of maslinic acid treatment on renal injury.
As shown in Fig. 4a and 4b, STZ induced mice showed an upregulation in the mass of glomerulus epithelium, thicker Bowman’s capsule, enhanced vacuolization in both the proximal and distal tubules, and much more arteriolopathy. On the other hand, 20 mg/kg maslinic acid treatment for 8 weeks protected the normal structure of glomeruli and tubules. 20 mg/kg maslinic acid treatment significantly ameliorated the renal injury induced by diabetes in mice.
Effects of maslinic acid treatment on renal oxidative stress.
To further investigate the mechanisms for maslinic acid to inhibit the renal injury induced by STZ, the oxidative biomarkers including MDA, ROS, MnSOD, GSH and CAT were measured. As shown in Fig. 5a and b, 20 mg/kg maslinic acid treatment for 8 weeks induced the down-regulation of MDA and ROS levels in renal tissues in diabetic mice, suggesting that maslinic acid decreased the oxidative stress in the kidney. Congruously, 20 mg/kg maslinic acid treatment upregulated the activities of enzymes targeting reactive oxygen species, including MnSOD, GSH and CAT (Fig. 5c, d and e). Thus, one of the possible mechanisms by which maslinic acid protected the renal tissue of diabetic mice was to reduce the oxidative pressure of the kidney.
Effects of maslinic acid treatment on renal inflammation.
The inflammatory factors including IL-6, MCP-1 and TNF-α in renual tissue and serum of mice in different groups was evaluated by ELISA and qRT-PCR assay. The renal IL-6, MCP-1 and TNF-α increased with STZ induction, and 20 mg/kg maslinic acid treatment lowered these pro-inflammatory cytokines in renal tissues dramatically (Fig. 6a, b and c). Congruously, the circulating levels of pro-inflammatory cytokines including IL-6, MCP-1 and TNF-α were also decreased with the treatment of maslinic acid in diabetic mice (Fig. 6d, e and f), suggesting that maslinic acid inhibited the inflammation during the progression of diabetic nephropathy.
Maslinic acid treatment activated renal AMPK/SIRT1 signaling pathway.
To further demonstrate the molecular mechanisms of the nephropathy effect of maslinic acid, Western-blotting assay was performed. As shown in Fig. 7a and b, maslinic acid treatment induced the phosphorylation of AMPK. The activation of AMPK thus induced the upregulation of SIRT1 (Fig. 7a and c). Since AMPK and SIRT1 play crucial roles in the oxidative protection and anti-inflammatory activity, it is possible that maslinic acid protected the renal function in diabetic mice via activating the AMPK/SIRT1 signaling pathway.