Comprehensive Effects of Suppression of MicroRNA-383 in Human Bone-Marrow-Derived Mesenchymal Stem Cells on Treating Spinal Cord Injury.

BACKGROUND/AIMS: Transplantation of bone-marrow-derived mesenchymal stem cells (MSCs) promotes neural cell regeneration after spinal cord injury (SCI). Recently, we showed that suppression of microRNA-383 (miR-383) in MSCs increased the protein levels of glial cell line derived neurotrophic factor (GDNF), resulting in improved therapeutic effects on SCI. However, the overall effects of miR-383 suppression in MSCs on SCI therapy were not determined yet. Here, we addressed this question. METHODS: We used bioinformatics tools to predict all miR-383-targeting genes, confirmed the functional bindings in a dual luciferase reporter assay. The effects of alteration of candidate genes in MSCs on cell proliferation were analyzed by MTT assay and by Western blotting for PCNA. The effects on angiogenesis were assessed by HUVEC assay. The effects on SCI in vivo were analyzed by transplantation of the modified MSCs into nude rats that underwent SCI. RESULTS: Suppression of miR-383 in MSCs not only upregulated GDNF protein, but also increased vascular endothelial growth factor A (VEGF-A) and cyclin-dependent kinase 19 (CDK19), two other miR-383 targets. MiR-383-suppression-induced increases in CDK19 resulted in a slight but significant increase in MSC proliferation, while miR-383-suppression-induced increases in VEGF-A resulted in a slight but significant increase in MSC-mediated angiogenesis. CONCLUSIONS: Upregulation of CDK19 and VEGF-A by miR-383 suppression in MSCs further improve the therapeutic potential of MSCs in treating SCI in rats.

Suppression of MicroRNA-383 Enhances Therapeutic Potential of Human Bone-Marrow-Derived Mesenchymal Stem Cells in Treating Spinal Cord Injury via GDNF.

BACKGROUND/AIMS: Transplantation of bone-marrow-derived mesenchymal stem cells (MSCs) has been used to treat spinal cord injury (SCI) to enhance tissue repair and neural cell regeneration. Glial cell line derived neurotrophic factor (GDNF) is an identified neural growth and survival factor. Here, we examined whether modification of GDNF levels in MSCs may further increase the potential of MSCs in promoting neural cell regeneration and subsequently the therapeutic outcome. METHODS: We examined the mRNA and protein levels of GDNF in human MSCs by RT-qPCR and Western blot, respectively. Bioinformatics analyses were done to predict microRNAs (miRNAs) that target GDNF in MSCs. The functional binding of miRNAs to GDNF mRNA was examined by a dual luciferase reporter assay. MSCs were transduced with adeno-associated virus (AAV) carrying null or antisense for miR-383 (as-miR-383), which were transplanted into nude rats that underwent SCI. The intact tissue, cavity volume, and recovery of locomotor activity were assessed. RESULTS: MSCs expressed very low GDNF protein, but surprisingly high levels of GDNF mRNA. Bioinformatics analyses showed that miR-383 inhibited protein translation of GDNF, through binding to the 3'-UTR of the GDNF mRNA. MSCs transduced with AAV-as-miR-383 further increased the intact tissue percentage, decreased cavity volume, and enhanced the recovery of locomotor activity in nude rats that underwent SCI, compared to MSCs. CONCLUSIONS: Suppression of miR-383 may increase the therapeutic potential of human bone-marrow-derived MSCs in treating SCI via augmentation of GDNF protein levels.

[Effect of jiaotai pill on pancreatic fat accumulation and islet cell apoptosis in rats with type 2 diabetes].

In this study, the rat type 2 diabetes mellitus (T2DM) model was established through tail vein injection with low dose of streptozotocin (STZ) and high fat diet for 8 weeks, and then treated with Jiaotai Pill. The oral glucose tolerance test (OGTT), fasting serum insulin (FINS), free fatty acid(FFA) levels and blood lipid were assayed. HOMA-IR was calculated. Pancreatic pathology was performed. And pancreatic triglyceride (TG) content was examined by the lipid extraction method. Pancreatic islet cell apoptosis were detected by terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). According to the results, the model group showed abnormal OGTT, increased FINS, HOMA-IR, FFA, lipid disorder, obvious fat accumulation and significantly increased TG content in pancreatic tissues, and enhanced pancreatic islet cell apoptosis. Compared with the model group, the Jiaotai Pill group displayed improved OGTT, reduced FINS, HOMA-IR, FFA, recovered lipid disorder, decreased fat accumulation and significantly declined TG content in pancreatic tissues, and lowered pancreatic islet cell apoptosis. In summary, Jiaotai pill could effectively treat type 2 diabetes in rats. Its mechanism may be related to the reduction in pancreatic fat accumulation and islet cell apoptosis.

CircATRNL1 protects against osteoarthritis by targeting miR-153-3p and KLF5.

BACKGROUND: Osteoarthritis (OA) is characterized by chondrocyte injury. Circular RNAs (circRNAs) are involved in the pathogenesis of various diseases, including OA. The purpose of this study was to determine the potential role of circATRNL1 in OA pathology in vitro. METHODS: Human chondrocytes were isolated and treated with interleukin-1 beta (IL-1ß) to mimic OA in vitro. High-throughput RNA sequencing was performed to identify differentially expressed circRNAs, miRNAs and mRNAs between IL and 1ß-treated chondrocytes and normal chondrocytes. The expression of circATRNL1, miR-153-3p and KLF5 was measured using quantitative real-time polymerase chain reaction (qRT-PCR). For functional analyses, cell apoptosis was assessed using a flow cytometry assay. Extracellular matrix (ECM) degradation was monitored by measuring the levels of ECM-associated proteins by Western blot. The potential target miRNAs of circATRNL1 were screened by bioinformatics analysis and verified by dual-luciferase reporter assay. RESULTS: The expression of circATRNL1 was decreased in IL-1ß-treated chondrocytes. CircATRNL1 overexpression ameliorated cell apoptosis and ECM degradation, which were promoted by IL-1ß treatment. Mechanistic analysis revealed that circATRNL1 directly targeted miR-153-3p and that miR-153-3p could reverse the inhibitory effects of circATRNL1 overexpression on inflammatory responses, cell apoptosis and ECM degradation. KLF5 is a target of miR-153-3p. CONCLUSION: Taken together, the results in this study suggested that circATRNL1 might ameliorate the development and progression of OA through regulating miR-153-3p/KLF5 axis. Our study increased the understanding of circRNAs as therapeutic targets in the treatment of OA.

Berberine reverses free-fatty-acid-induced insulin resistance in 3T3-L1 adipocytes through targeting IKKbeta.

AIM: To investigate the effects and molecular mechanisms of berberine on improving insulin resistance induced by free fatty acids (FFAs) in 3T3-L1 adipocytes. METHODS: The model of insulin resistance in 3T3-L1 adipocytes was established by adding palmic acid (0.5 mmol/L) to the culture medium. Berberine treatment was performed at the same time. Glucose uptake rate was determined by the 2-deoxy-[(3)H]-D-glucose method. The levels of IkB kinase beta (IKKbeta) Ser(181) phosphorylation, insulin receptor substrate-1(IRS-1) Ser(307) phosphorylation, expression of IKKbeta, IRS-1, nuclear transcription factor kappaB p65 (NF-kappaB p65), phosphatidylinositol-3-kinase p85 (PI-3K p85) and glucose transporter 4 (GLUT4) proteins were detected by Western blotting. The distribution of NF-kappaB p65 proteins inside the adipocytes was observed through confocal laser scanning microscopy (CLSM). RESULTS: After the intervention of palmic acid for 24 h, the insulin-stimulated glucose transport in 3T3-L1 adipocytes was inhibited by 67%. Meanwhile, the expression of IRS-1 and PI-3K p85 protein was reduced, while the levels of IKKbeta Ser(181) and IRS-1 Ser(307) phosphorylation, and nuclear translocation of NF-kappaB p65 protein were increased. However, the above indexes, which indicated the existence of insulin resistance, were reversed by berberine although the expression of GLUT4, IKKbeta and total NF-kappaB p65 protein were not changed during this study. CONCLUSION: Insulin resistance induced by FFAs in 3T3-L1 adipocytes can be improved by berberine. Berberine reversed free-fatty-acid-induced insulin resistance in 3T3-L1 adipocytes through targeting IKKbeta.

Jiao-tai-wan Up-regulates Hypothalamic and Peripheral Circadian Clock Gene Cryptochrome and Activates PI3K/AKT Signaling in Partially Sleep-deprived Rats.

This study aims to explore the effect and mechanism of Jiao-tai-wan (JTW) on systemic and tissue-specific inflammation and insulin resistance in obesity-resistant (OR) rats with chronic partial sleep deprivation (PSD). OR rats with PSD were orally given JTW and Estazolam for 4 weeks. The amount of food intake and metabolic parameters such as body weight increase rate, fasting plasma glucose (FPG), fasting insulin (FINS), homeostasis model assessment-insulin resistance (HOMA-IR) and plasma inflammatory markers were measured. The expression levels of circadian proteins cryptochrome 1 (Cryl) and cryptochrome 2 (Cry2) in hypothalamus, adipose and liver tissues were also determined. Meanwhile, the mRNA expression of inflammatory markers, activity of nuclear factor kappa B (NF-κB) p65 protein, as well as the expression levels of insulin signaling pathway proteins in hypothalamus, adipose and liver tissues were measured. Additionally, cyclic adenosine 3', 5'-monophosphate (cAMP) and activity of vasodilator-stimulated phosphoprotein (VASP) in hypothalamus tissue were measured. JTW significantly decreased the body weight increase rate and food intake, ameliorated systemic inflammation and insulin resistance. JTW effectively ameliorated inflammation and increased PI3K/AKT signaling activation in hypothalamus, adipose and liver. Interestingly, all these changes were associated with the up-regulation of circadian gene Cryl and Cry2 protein expression. We also found that in hypothalamus tissue of PSD rats, down-regulation of Cryl and Cry2 activated cAMP/PKA signaling and then led to inflammation, while JTW inhibited this signaling. These results suggested that JTW has the beneficial effect on ameliorating inflammation and insulin resistance in partially sleep-deprived rats by up-regulating Cry expression.

[Effect of berberine on insulin secretion and glucokinase activity of NIT-1 cells].

To investigate the effect of berberine on insulin secretion of NIT-1 cells stimulated by glucose and the possible molecular mechanism, we used radioimmunoassay, scintillation counting technique, enzymatic method and Western blotting to measure the effects of berberine on insulin secretion, glucose utilization, the activity of glucokinase (GK) and protein level of GK and GK regulation protein (GKRP). Compared with untreated group, insulin secretion level, glucose utilization, the activity and protein level of GK in NIT-1 cells stimulated by high concentration of glucose were increased significantly in berberine group (P < 0.05), while the protein level of GKRP in berberine group decreased markedly. In conclusion, berberine can promote insulin secretion of NIT-1 cells induced by high concentration of glucose. The possible molecular mechanism may be associated with berberine acting as a GK activator, improving glucose utilization, enhancing the activity and protein expression level of GK, as well as decreased the protein level of GKRP.

Effect of huanglian jiedu decoction ) on glucose transporter 4 expression in adipose and skeletal muscle tissues of insulin resistant rats.

OBJECTIVE: To investigate the effects of Huanglian Jiedu Decoction (HLJDD) on glucose transporter 4 (GLUT4) protein expressions in insulin-resistant murine target tissues. METHODS: The experimental male Wistar rats were established into insulin resistant models by injecting streptozotocin (STZ 30 mg/kg) via caudal vein and feeding them with high fat high caloric diet, and randomly divided into the model group, the aspirin group and the HLJDD group. Besides, a normal group was set up for control. Changes of body weight (BW), levels of serum fasting blood glucose (FBG), serum fasting insulin (FINS) and oral glucose tolerance test (OGTT) were routinely determined. The expression of GLUT4 protein in adipose and skeletal muscle tissues before and after insulin stimulation was determined with Western blot. RESULTS: In the HLJDD group after treatment, BW and FBG got decreased, OGTT improved, and the expression and translocation of GLUT4 protein elevated obviously, either before or after insulin stimulation, as compared with those in the model group, showing significant differences respectively. CONCLUSION: The mechanism of improving insulin resistance by HLJDD is probably associated with its effect in elevating GLUT4 protein expression and translocation in adipose and skeletal muscle tissues of insulin resistant rats.

Effect of Jiaotai Pill () on intestinal damage in partially sleep deprived rats.

OBJECTIVE: To explore the effect and mechanism of Jiaotai Pill (, JTW) on intestinal mucosal damage in rats with chronic partial sleep deprivation (PSD). METHODS: Obesity resistant (OR) rats were selected, and underwent 4 h PSD by being exposed to environmental noise for 4 weeks. During the whole PSD period, JTW and estazolam were orally given to the rats respectively in the treating groups. Plasma concentration of lipopolysaccharide (LPS) which is the marker of gut-origin endotoxemia was examined. Intestinal morphology changes were observed by optical microscopy. The protein expression of occludin (Ocln) in the intestine was measured by immunofluorescence technique and Western blot. The expressions of circadian proteins cryptochromes (Cry1 and Cry2) in the intestine were also determined. RESULTS: The treatment of JTW significantly decreased LPS level in OR rats with PSD (P<0.05). JTW also attenuated insomnia-induced intestinal injury like shorter, sparse and incomplete villus, wide gap between the villus, mucosal swelling and congesting (P<0.05). These changes were associated with the effect of JTW on up-regulating the expressions of Cry1 protein, Cry2 protein and Ocln protein in the intestine. CONCLUSIONS: JTW has the beneficial effect on improving intestinal mucosal damage caused by PSD. The mechanism appears to be related to the modulation of the expressions of circadian proteins and Ocln protein in the intestine, thereby attenuating inflammation and improving insulin resistance in insomnia rats.

Berberine relieves insulin resistance via the cholinergic anti-inflammatory pathway in HepG2 cells.

Berberine (BBR) is an isoquinoline alkaloid extracted from Rhizoma coptidis and has been used for treating type 2 diabetes mellitus (T2DM) in China. The development of T2DM is often associated with insulin resistance and impaired glucose uptake in peripheral tissues. In this study, we examined whether BBR attenuated glucose uptake dysfunction through the cholinergic anti-inflammatory pathway in HepG2 cells. Cellular glucose uptake, quantified by the 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-2-deoxy-D-glucose (2-NBDG), was inhibited by 21% after HepG2 cells were incubated with insulin (10(-6) mol/L) for 36 h. Meanwhile, the expression of alpha7 nicotinic acetylcholine receptor (α7nAChR) protein was reduced without the change of acetylcholinesterase (AChE) activity. The level of interleukin-6 (IL-6) in the culture supernatant, the ratio of phosphorylated I-kappa-B kinase-ß (IKκß) Ser181/IKKß and the expression of nuclear factor-kappa B (NF-κB) p65 protein were also increased. However, the treatment with BBR enhanced the glucose uptake, increased the expression of α7nAChR protein and inhibited AChE activity. These changes were also accompanied with the decrease of the ratio of pIKKß Ser181/IKKß, NF-κB p65 expression and IL-6 level. Taken together, these results suggest that BBR could enhance glucose uptake, and relieve insulin resistance and inflammation in HepG2 cells. The mechanism may be related to the cholinergic anti-inflammatory pathway and the inhibition of AChE activity.

Effects of berberine and cinnamic acid on palmitic acid-induced intracellular triglyceride accumulation in NIT-1 pancreatic ß cells.

OBJECTIVE: To investigate the effects of berberine (BBR) and cinnamic acid (CA), the main active components in Jiaotai Pill (, JTP), on palmitic acid (PA)-induced intracellular triglyceride (TG) accumulation in NIT-1 pancreatic ß cells. METHODS: Cells were incubated in culture medium containing PA (0.25 mmol/L) for 24 h. Then treatments with BBR (10 µmol/L), CA (100 µmol/L) and the combination of BBR and CA (BBR+CA) were performed respectively. Intracellular lipid accumulation was assessed by Oil Red O staining and TG content was measured by colorimetric assay. The expression of adenosine monophosphate-activated protein kinase (AMPK) protein and its downstream lipogenic and fatty acid oxidation genes, including fatty acid synthase (FAS), acetyl-coA carboxylase (ACC), phosphorylation acetyl-coA carboxylase (pACC), carnitine acyl transferase 1 (CPT-1) and sterol regulating element binding protein 1c (SREBP-1c) were determined by Western blot or real time polymerase chain reaction. RESULTS: PA induced an obvious lipid accumulation and a significant increase in intracellular TG content in NIT-1 cells. PA also induced a remarkable decrease in AMPK protein expression and its downstream targets such as pACC and CPT-1. Meanwhile, AMPK downstream lipogenic genes including SREBP-1c mRNA, FAS and ACC protein expressions were increased. Treatments with BBR and BBR+CA, superior to CA, significantly reversed the above genes changes in NIT-1 pancreatic ß cells. However, the synergistic effect of BBR and CA on intracellular TG content was not observed in the present study. CONCLUSION: It can be concluded that in vitro, BBR and BBR+CA could inhibit PA-induced lipid accumulation by decreasing lipogenesis and increasing lipid oxidation in NIT-1 pancreatic ß cells.

Effects of emodin on treating murine nonalcoholic fatty liver induced by high caloric laboratory chaw.

AIM: To investigate the effects of emodin on the treatment of non-alcoholic fatty liver in rats induced by high caloric laboratory chaw. METHODS: Non-alcoholic fatty liver model was successfully established by feeding with high caloric laboratory chaw for 12 wk. Then the model rats were randomly divided into 3 groups, namely model control group, emodin group and dietary treatment group. The rats in emodin group were given emodin at dose of 40 mg/(kg x d) while animals in other groups were given distilled water of the same volume. The rats in model control group were fed with high caloric laboratory chaw while animals in other groups were fed with normal diet. Four weeks later, liver index (liver/body weight ratio), serum activities of liver-associated enzymes, blood lipid, fasting blood glucose, fasting plasma insulin, HOMA insulin resistance index (HOMA-IR), hepatic triglyceride content and histology features of all groups were assayed. The expression of hepatic peroxisomal proliferator activated receptor (PPAR) gamma was determined by RT-PCR. RESULTS: The body weight, liver index, serum activities of alanine aminotransferase (ALT), blood lipid, hepatic triglyceride content of model control group were significantly elevated, with moderate to severe hepatocyte steatosis. The expression of hepatic PPAR gamma mRNA was obviously reduced in model control group. Compared with model control group, the body weight, liver index, serum activities of ALT, blood lipids and hepatic triglyceride of emodin group significantly decreased and hepatic histology display was also greatly improved. Meanwhile, the expression of hepatic PPAR gamma mRNA was elevated. However, high serum activities of ALT and hyperlipidemia were persisted in dietary treatment group although liver index was decreased and liver histology was somewhat improved. CONCLUSION: It is suggested that emodin might be effective in the treatment of non-alcoholic fatty liver in rats. Its therapeutic mechanism could be associated with increasing the expression of hepatic PPAR gamma mRNA.

[Study on therapeutic effects of metformin on rat fatty livers induced by high fat feeding].

OBJECTIVES: To explore the therapeutic effects of metformin on rat fatty livers induced by high fat feeding. METHODS: A fatty liver model was established by feeding rats with a high caloric laboratory chow for 12 weeks, then the rats were randomly divided into three groups, i.e. model control group, metformin group and dietary treatment group. A normal control group was organized at the same time. The rats of the metformin group were given metformin 156 mg/kg/d while the other groups were given distilled water of the same volume by stomach feeding. The model control group rats were fed with high caloric laboratory chow while other groups were fed a normal diet. After four weeks, all the animals were sacrificed. Liver index (liver/body weight ratio), serum activities of liver-associated enzymes, blood lipids, liver triglycerides, fasting blood glucose, fasting plasma insulin, HOMA insulin resistance index (HOMA-IR), and the liver histology of rats of all groups were assayed. RESULTS: The body weight, liver index, serum activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), total cholesterol (TC), triglycerides (TG) and liver triglycerides in the model group increased significantly, while HDL-cholesterol concentration decreased significantly. Fasting blood glucose, fasting plasma insulin and HOMA-IR showed an increasing tendency, but there was no significant difference of those indexes among the three groups. The liver histology in the model group showed moderate to severe steatosis, mainly as macro vesicle steatosis, lobular inflammatory, cell infiltration and necrosis. Compared with the model group, the levels of body weight, liver index, serum ALT, ALP, TC, TG and liver triglycerides in the metformin group were significantly lower and were similar to those of the normal group, while their HDL-cholesterol concentration was significantly higher. The liver histology in the metformin group was nearly normal. In the dietary treatment group, hyperlipidemia persisted, although liver index and GGT were lower and the liver histology changes were somewhat milder. CONCLUSION: It is suggestive that metformin might be effective in treating rat fatty liver induced by high fat feeding.

[Protective effects of Huanglian Jiedu decoction on vascular endothelial function in type 2 diabetic rats].

OBJECTIVE: To investigate the effects of Huanglian Jiedu (HLJD) decoction on vascular endothelial function in type 2 diabetic rats and explore the prophylactic and therapeutic significance and pharmacological mechanism of HLJD decoction in type 2 diabetic angiopathic complication. METHOD: The murine type 2 diabetes models were induced by the intravenous injection of a small dose of streptozotocin plus high fat and high caloric laboratory chow. Then modeled diabetic animals were divided into model group, HLJD group, and aspirin group. Normal ratsfed with routine chow were designated as normal group. The oral glucose tolerance test (OGTT) were performed in all animals, 9 weeks after treatment, the changes of murine body weights and levels of fasting blood glucose (FBG), serum total cholesterol (TC), triglycerides (TG), high density lipoprotein-cholesterol(HDL-C), fasting serum insulin (FINS), serum nitric oxide, plasma endothelin, angiotensin II and von Willebrand Factor (vWF) were determined 10 weeks after treatment. RESULT: Compared with model group, the result of OGTT of HLJD group was improved. The levels of the body weights, TC, TG and ET in HLJD group weredecreased (P < 0.05). The levels of FBG,INS, AngII and vWFwere significantly decreased (P < 0.01), and the levels of HDL-C and NO were obviously increased (P < 0.05), as compared with those in model group. Furthermore. The levels of FBG was lower in HLJD group than in aspirin group (P < 0.05), and the improvement of TG, HDL-C,NO, AngII, vWF levels in HLJD group was more greatly than that in aspirin group, but there was not significant difference between two groups (P > 0.05). CONCLUSION: It is suggested by the present results that HLJD decoction could protect vascular endothelium from early damage in type 2 diabetes. The protective effects of HLJD on endothelium might be related to its ability of reducing the blood glucose, adjusting plasma lipids profiles, improving insulin resistance, antagonizing inflammatory mediators and inducing endothelium-dependent vascular relaxation.

Berberine inhibits hepatic gluconeogenesis via the LKB1-AMPK-TORC2 signaling pathway in streptozotocin-induced diabetic rats.

AIM: To investigate the molecular mechanisms of berberine inhibition of hepatic gluconeogenesis in a diabetic rat model. METHODS: The 40 rats were randomly divided into five groups. One group was selected as the normal group. In the remaining groups (n = 8 each), the rats were fed on a high-fat diet for 1 mo and received intravenous injection of streptozotocin for induction of the diabetic models. Berberine (156 mg/kg per day) (berberine group) or metformin (184 mg/kg per day) (metformin group) was intragastrically administered to the diabetic rats and 5-aminoimidazole-4-carboxamide1-ß-D-ribofuranoside (AICAR) (0.5 mg/kg per day) (AICAR group) was subcutaneously injected to the diabetic rats for 12 wk. The remaining eight diabetic rats served as the model group. Fasting plasma glucose and insulin levels as well as lipid profile were tested. The expressions of proteins were examined by western blotting. The nuclear translocation of CREB-regulated transcription co-activator (TORC)2 was observed by immunohistochemical staining. RESULTS: Berberine improved impaired glucose tolerance and decreased plasma hyperlipidemia. Moreover, berberine decreased fasting plasma insulin and homeostasis model assessment of insulin resistance (HOMA-IR). Berberine upregulated protein expression of liver kinase (LK)B1, AMP-activated protein kinase (AMPK) and phosphorylated AMPK (p-AMPK). The level of phophorylated TORC2 (p-TORC2) protein in the cytoplasm was higher in the berberine group than in the model group, and no significant difference in total TORC2 protein level was observed. Immunohistochemical staining revealed that more TORC2 was localized in the cytoplasm of the berberine group than in the model group. Moreover, berberine treatment downregulated protein expression of the key gluconeogenic enzymes (phosphoenolpyruvate carboxykinase and glucose-6-phosphatase) in the liver tissues. CONCLUSION: Our findings revealed that berberine inhibited hepatic gluconeogenesis via the regulation of the LKB1-AMPK-TORC2 signaling pathway.

Fenugreek lactone attenuates palmitate-induced apoptosis and dysfunction in pancreatic ß-cells.

AIM: To investigate the effect of fenugreek lactone (FL) on palmitate (PA)-induced apoptosis and dysfunction in insulin secretion in pancreatic NIT-1 ß-cells. METHODS: Cells were cultured in the presence or absence of FL and PA (0.25 mmol/L) for 48 h. Then, lipid droplets in NIT-1 cells were observed by oil red O staining, and the intracellular triglyceride content was measured by colorimetric assay. The insulin content in the supernatant was determined using an insulin radio-immunoassay. Oxidative stress-associated parameters, including total superoxide dismutase, glutathione peroxidase and catalase activity and malondialdehyde levels in the suspensions were also examined. The expression of upstream regulators of oxidative stress, such as protein kinase C-α (PKC-α), phospho-PKC-α and P47phox, were determined by Western blot analysis and real-time PCR. In addition, apoptosis was evaluated in NIT-1 cells by flow cytometry assays and caspase-3 viability assays. RESULTS: Our results indicated that compared to the control group, PA induced an increase in lipid accumulation and apoptosis and a decrease in insulin secretion in NIT-1 cells. Oxidative stress in NIT-1 cells was activated after 48 h of exposure to PA. However, FL reversed the above changes. These effects were accompanied by the inhibition of PKC-α, phospho-PKC-α and P47phox expression and the activation of caspase-3. CONCLUSION: FL attenuates PA-induced apoptosis and insulin secretion dysfunction in NIT-1 pancreatic ß-cells. The mechanism for this action may be associated with improvements in levels of oxidative stress.

Inhibition of proprotein convertase subtilisin/kexin type 9: a novel mechanism of berberine and 8-hydroxy dihydroberberine against hyperlipidemia.

OBJECTIVE: To investigate the effect and molecular mechanisms of different doses of 8-hydroxy dihydroberberine (Hdber) for the treatment of hyperlipidemia in rats. METHODS: A rat model of hyperlipidemia was established by feeding rats a high-fat diet for 4 weeks in 70 rats of 80 animals, and 10 rats were randomly selected as control group. The hyperlipidemic rats were then randomly divided into the following groups: a model group (MOD); a berberine group [BBR, 156 mg/(kg day)]; Hdber groups, which were treated with different doses of Hdber [78, 39 and 19.5 mg/(kg day)]; and a simvastatin group [SIM, 4 mg/(kg day)]. The corresponding therapy was administered to the rats of each treatment via gastric tubes. Normal animals were used as a control group. The blood levels of various lipids, including total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, free fatty acid (FFA), apolipoprotein AI(Apo-AI) and apolipoprotein B (Apo-B) were examined. The protein expressions of low-density lipoprotein receptor (LDL-R), sterol regulatory element-binding protein 2 (SREBP-2), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and proprotein convertase subtilisin/kexin type 9 (PCSK-9) in liver tissues were determined by Western blot analysis. RESULTS: Compared with the control group of rats, the model group demonstrated a deteriorated blood lipid profile and exhibited increased expression levels of PCSK-9 protein in their liver tissues (P<0.01). In addition, the high-fat diet decreased the expression levels of LDL-R, SREBP-2 and HMGCR proteins in murine liver tissues. However, the addition of berberine or Hdber reversed the blood lipid profile changes (P<0.05 or P<0.01), decreased the expression levels of PCSK-9 proteins (P<0.01), and increased the expression levels of LDL-R proteins in the hyperlipidemic rats (P<0.01). These compounds did not significantly influence the expression levels of SREBP-2 and HMGCR proteins in the hyperlipidemic rats. CONCLUSIONS: Hdber is effective in the treatment of hyperlipidemia in rats. The therapeutic mechanisms of Hdber may be associated with increasing the expression of LDL-R protein and decreasing the expression of PCSK-9 protein in liver tissues.