Ⅰ, Berberine Upregulation Of Liver Low Density Lipoprotein Receptor MRNA Expression Ⅱ, Berberine Analogue Y53 In Diabetic Mice In Vivo Anti-inflammatory, Anti-oxidation And Prevention Of Diabetic Nephropathy

Our previous studies showed that natural product berberine (BBR) lowered serum cholesterol (CHO) by increasing liver low-density lipoprotein receptor (LDLR) mRNA expression in an extracellular signal-regulated kinase (ERK)-dependent manner. However, how the ERK pathway was activated by BBR was not clear. This work was designed to explore the upstream cellular signaling molecules recruited by BBR to activate the ERK mitogen-activated protein kinase (MAPK) cascade and to study the related mechanisms. In the blocking experiments, multiple chemical inhibitors such as GW5074, a specific inhibitor of Raf-1; manumycin A, a Ras inhibitor; staurosporine, an inhibitor of protein kinase C (PKC); and compound C, a specific inhibitor of AMP-activated protein kinase (AMPK) were used. Specific small interfering RNAs (siRNAs) were also used to silence the expression of AMPK. Western blot was used to determine the phosphorylation of protein kinases; quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) was used to determine the expression level of the LDLR mRNA. The influence of BBR on related signaling pathways in vivo was studied in hyperlipidemic rats induced by fat emulsion-feeding. In the in vitro experiments, the results indicated that BBR increased the phosphorylation of Raf-1 at serine 338 (ser338) significantly in HL-7702 cells and HepG2 cells. The stimulating efficacy of BBR on Raf-1 was time-dependent and dose-dependent. BBR had no influence on Ras activity; its stimulating activities on Raf-1 signaling and LDLR mRNA expression were blocked by GW5074 completely, but were not influenced by manumycin A, a Ras inhibitor. In the animal experiment, BBR activated hepatic Raf-1 signaling and up-regulated LDLR mRNA expression in a rat model of hyperlipidemia with no impact on liver Ras activity. Importantly, our results showed that the stimulating activities of BBR on hepatic Raf-1 signaling and LDLR mRNA expression were totally blocked by compound C, a specific inhibitor of AMPK, and also by silencing its expression with siRNA. Taken together, our results demonstrate for the first time that BBR up-regulates LDLR mRNA expression through Ras-independent, but AMPK-dependent Raf-1 activation in liver cells. Our study will help to elucidate the molecular pharmacology of BBR in CHO reduction and provide new scientific evidence for its clinical application to treat hypercholesterolemia.This study was designed to investigate the antioxidant, anti-inflammatory and diabetic nephropathy (DN)-preventing activities of pseudoberberine (Y53), a berberine (BBR) analogue, in diabetic mice.Firstly, the influences of Y53 and BBR on blood glucose, liver and kidney functions were observed in normal mice. The Kunming (KM) mice were used in the experiment, Y53 or BBR at a dose of 100 mg/kg was orally administered to the animals for 30 days. The general state of the mice were observed, the fasting blood glucose (FBG) level, liver and kidney function parameters were determined after the experiment. In addition, livers and kidneys of the mice were harvested for pathological examination by hematoxylin & eosin (H&E) staining. Our results indicated that the mice remained healthy after the administration of Y53 or BBR. At a dose of 100 mg/kg, Y53 or BBR had no influences on FBG, body weight, liver and kidney functions of the KM mice. The results of the pathological examination also showed that Y53 and BBR had no significant toxicity to the liver and kidney of the mice.Then, the anti-oxidative stress, anti-inflammatory and glucose/lipid metabolism modulating activities and mechanisms of Y53 were studied in type 1 diabetic mice with fatty liver. Diabetes mellitus (DM) of the C57BL/6J mice was induced by high fat diet (HFD)-feeding followed by single dose intraperitoneal injection of streptozotocin (STZ) (120 mg/kg). The animals with DM were divided into 4 groups, which were treated with saline,50 mg/kg of BBR,50 mg/kg of Y53 or 100 mg/kg of BBR, respectively. The results showed that Y53 potently lowered serum lipids and FBG, increased serum insulin and pancreas weight and up-regulated hepatic expression of low-density lipoprotein receptor (LDLR) and insulin receptor (InsR) in diabetic mice. The Y53 also suppressed liver steatosis, reduced fat accumulation, liver weight and index and restored liver function in the mice. The mice developed obvious oxidative stress and proinflammatory response after the onset of DM. Y53 significantly increased superoxide dismutase (SOD) activity and reduced malondialdehyde (MDA) level in the serum, liver and pancreas. On the other hand, Y53 greatly reduced the mRNA expression levels of proinflammatory cytokines like interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a) but increased those of nuclear factor erythroid-2-related factor-2 (Nrf2), heme oxygenase-1 (HO-1) and NADPH quinine oxidoreductase-1 (NQO-1) in the liver and pancreas of the mice. The efficacies of Y53 were superior to those of BBR at the same dose and close to those of BBR at double dose.In another animal experiment, we studied the preventive effects of Y53 on DN. Type 1 DM of the C57BL/6J mice was induced by intraperitoneal injection of STZ at 120 mg/kg. The diabetic animals were treated with saline,50 mg/kg of BBR,50 mg/kg of Y53 or 5 mg/kg of rosiglitazone (ROSI), respectively. The parameters of blood, serum, urine and kidney were determined by kits; kidneys of the mice were subjected to H&E staining; renal mRNA expression levels of target gene were determined by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). Our results showed that Y53 greatly reduced the FBG and glycosylated hemoglobin (GHb), improved diabetic symptoms such as polyphagia and polyuria in the diabetic mice. Y53 potently reduced the serum blood urea nitrogen (BUN), serum creatinine (Scr),24 h urinary protein, kidney index, serum and renal advanced glycation end-products (AGEs) and nitric oxide (NO), renal MDA/cholesterol (CHO)/triglyceride (TG), but increased renal SOD activity. In the pathological examination, Y53 greatly restored renal morphology and suppressed glomerular sclerosis. Y53 significantly reduced the renal expression of proinflammatory cytokines and genes critical for renal fibrosis such as the transforming growth factor-β (TGF-β) and smad2. In this experiment, the renoprotective efficacies of Y53 were superior to those of BBR and ROSI.In summary of the above results, Y53 has no influence on FBG in normal mice, but can improve the metabolisms of glucose and lipids greatly in mice with DM. In addition, Y53 can ameliorate oxidative stress and proinflammatory response, prevent the development of DN and protect renal function significantly in diabetic mice. We suggest that in the future, Y53 may be developed as a novel class of oral hypoglycemic agent for the treatment of DM as well as its complications such as the DN.