Renoprotective Effect Of PKC Inhibitor Breviscapine And Its Mechanism In Experimental Diabetic Rats

Background and objective The distinctive pathological changes in the early stage of diabetic nephropathy is characterized by glomerular hypertrophy s extracellular matrix (ECM) accumulation and glomerular basement membrane thickening and most result in glomerulosclerosis gradually, further induce end-stage renal disease (ESRD).The progress of DN is the result of interaction between glucose metabolic disorder and turbulance in blood hydrodynamics, its downstream mechanism involved oxidation stress , protein kinase C (PKC) and inflammation course . The key NADPH oxidation enzyme in the course of oxidation stress is activated through a PKC dependent pathway, on the other hand, reactive oxygen species (ROS) and PKC are also important upstream activator in the inflammation course of DN development, and the recent study in vivo and in vitro evidence demonstrated that antioxidant , PKC inhibitor and anti-inflammatory treatment have significant protective effects in DN. Breviscapine is extracted from a Chinese herb Erigeron breviscapus (Vant), Hand Mazz. The active ingredient is 4'- OH- scutellarin- 7-o- glucuronide and possesses a variety of pharmacological functions beyond their hemodynamic effects such as dilating micranium, reducing blood viscosity, and ameliorating microcirculation, especially as anti-oxidative stress agent and PKC inhibitor. Ruboxistaurin (LY333531) mesylate is a bisindolylmaleimide that shows a high degree of specificity for inhibiting pkc isoforms and great experimental study evidence indicates that LY333531 have distinct renal protective effects, the purpose of the present study was to investigate the possible renal protective effects of breviscapine and LY333531 on macrophage recruitment, oxidative stress\ PKC activation and expression of MCP-1, ICAM-1, TGFβ₁ andCTGF in renal in diabetic rats induced by STZ. Methods Fourty adult male Sprague-Dawley rats were separated into four groups at random. Control group (n=10), model group (n=10). model group treated with breviscapine (breviscapine group. n=10) and model group treated with LY333531 (LY333531 group, n=10). To induce an experimental model of diabetes, rats received a single intraperitoneal injection of STZ (60mg?kg^'). Breviscapine group received breviscapine 20mg?kg^'?d¹ by gavage, LY333531 group received LY333531 10mg-kg^'*d^I by gavage. 8 weeks after STZ injection, the following determinations were done in samples: (1) plasma glucose (BG) were determined according to standard methods; (2) renal tissue malondiadehyde (MDA)> superoxide diamutase (SOD^ catalase (CAT) and glutathione peroxidase (GSH-Px) were determined by spectrophotometric method. 24 hours albumin excretion rate (AER) were measured by enzyme immunoassay (EIA). PKC activity were measuared by y-ATP radioactive penetration. Renal lesions were evaluated using PAS taining; the mean glomerular cross-section area (Ag^ glomerular volume (Vra), mesangial area (Am) were measured by using pathology image analysis systemsoftware. Immunohistochemistry for ED-1 (macrophages marker), MCP-1 ^ ICAM-1 > TGFPi and CTGF were performed by streptavidin-biotin complex (SABC) technique. Results 1. BG, Body weight (BW) , kidney weight (KW) and ratio of KW to BW (KW/BW): 8 weeks after STZ injection, there was a significant increase in fasting BG ( PO.01) and BW was significantly decreased (p<0.05) in diabetic rats compared with control group. The BG level of breviscapine and LY333531 group have no statistically significant difference compared with diabetes group, but BW of the group of LY333531 is significantly higher than diabetes group(.P<0.05). KW and KW/BW in breviscapine and LY333531 group were significantly lower than diabetic groupC^O.05). 2. AER : AER in diabetes group were significantly higher than control group (P<0.05. P<0.01) and AER in breviscapine and LY333531 group were significantly lower than diabetic group (PO.05) . 3. Renal pathologic morphology: Compared with control, AG^VG n Am of diabetes group were significantly increased and increased Aq CAT and GSH-Px activity in renal tissue: MDA level in renal tissue and urine in diabetic rats were higher than those of the control group and SOD, CAT, GSH-PX activity were significantly reduced in the renal of the diabetic grourj compared with control value. Elevated MDA level in renal tissue and urine as well as decreased SOD, CAT, GSH-PX activity in renal tissue were remitted by breviscapine and LY333531 (.PO.05) . Further more MDA level in renal tissue and urine in breviscapine group were significantly lower than the LY333531 group (PO.05) , while GSH-PX activity in breviscapine group were significantly higher than it of the LY333531 group (PO.05) . 5. PKC activity : Compared with control. PKCu PKCc, PKCm and PKCm/PKCc activity of diabetes group were significantly increased (P O.05, PO.01), and increased PKCts PKCcn PKCm > PKCm/PKCc were significantly attenuated by treatment with breviscapine and LY333531 (PO.05) . 6. Macrophages infiltration in renal tissue : Renal from diabetic group presented macrophages (ED-1 positive cell) significant infiltration compared with the control (PO.01), and macrophages infiltration in breviscapine or LY333531 group all significantly lower than diabetic group (PO.01), but macrophages infiltration in LY333531 group are also significantly higher than the control (P O.01). 7. MCP-1 and ICAM-1 expression in renal tissue : Compared with control, MCP-1 and ICAM-1 expression were significantly increased in diabetic rats (PO.01), while MCP-1 and ICAM-1 expression in breviscapine or LY333531 group were significantly lower than diabetic group (PO.05). 8. TGFpi and CTGF expression in renal tissue : TGFp, and CTGF immunostaining were found in greatest abundance in the renal even in tubule-interstitium of diabetic group compared with control (PO.01) , at the same time in breviscapine or LY333531 group TGF|3i and CTGF expression were significantly lower than diabetic group (PO.05). Conclusion Renoprotective...