Inhibitory Mechanism of Epigallocatechin Gallate on Fibrillation and Aggregation of Amidated Human Islet Amyloid Polypeptide.

The abnormal fibrillation of human islet amyloid polypeptide (hIAPP) is associated with development of type II diabetes mellitus (T2DM). (-)-Epigallocatechin gallate (EGCG) can bind amyloid proteins to inhibit the fibrillation of these proteins. However, the mechanic detail of EGCG inhibiting amyloid formation is still unclear at the molecular level. In the present work, we sought to investigate the effect of EGCG on amidated hIAPP (hIAPP-NH2 ) fibrillation and aggregation by using spectroscopic and microscopic techniques, and also sought to gain insights into the interaction of EGCG and hIAPP22-27 by using spectroscopic experiments and quantum chemical calculations. ThT fluorescence, real-time NMR, and TEM studies demonstrated that EGCG inhibits the formation of hIAPP-NH2 fibrils, while promoting the formation of hIAPP-NH2 amorphous aggregates. Phenylalanine intrinsic fluorescence and NMR studies of the EGCG/hIAPP22-27 complex revealed three important binding sites including the A ring of EGCG, residue Phe23, and residue Ile26. DFT calculations identified the dominant binding structures of EGCG/Phe23 and EGCG/Ile26 complexes, named structure I and structure II, respectively. Our study demonstrates the inhibitory mechanism of EGCG on fibrillation and aggregation of hIAPP-NH2 in which EGCG interacts with hIAPP-NH2 through hydrogen bonding and π-π interactions between the A ring and residue Phe23 as well as hydrophobic interactions between the A ring and residue Ile26, which can thus inhibit the interpeptide interaction between hIAPP-NH2 monomers and finally inhibit fibrillation of hIAPP-NH2 . This study agrees with and reinforces previous studies and offers an intuitive explanation at both the atomic and molecular levels. Our findings may provide an invaluable reference for the future development of new drugs in the management of diabetes.

[Treating Type 2 Diabetes Mellitus Patients Complicated with Metabolic Syndrome by Benefiting Qi Dissolving Method].

Objective To observe clinical efficacy of Yiqi Huaju Recipe (YHR) combined routine Western medical treatment on type 2 diabetes mellitus (T2DM) patients complicated metabolic syndrome (MetS). Methods Totally 96 T2DM patients complicated MetS were assigned to the treatment group (YHR +routine Western drugs) and the control group (placebo +routine Western drugs) according to random digit table, 48 cases in each group. The therapeutic course for all was 12 weeks. Body mass index (BMI) , waistline, waist-hip ratio (WHR) , fasting plasma glucose (FPG) , 2 h postprandial plasma glucose (2 h PPG) , glycosylated hemoglobin A1c (HbAlc) , homeostasis model assessment of insulin resistance (HOMA-IR) , blood lipids, blood pressure, disease transformation of MetS, changes of con- stituent numbers were detected before and after treatment. Results BMI, WHR, waistline obviously decreased in the treatment group after treatment, with statistical difference as compared with the control group (P<0.01 , P <0.05). Post-treatment FPG, 2 h PPG, HbAlc, HOMA-IR, systolic blood pressure (SBP), diastolic blood pressure (DBP) , and mean artery pressure (MAP) obviously decreased in the two groups, but more obviously in the treatment group (P <0. 05). Post-treatment total cholesterol (TC) , low density lipoprotein cholesterol (LDL-C) , and triglycerides (TG) all obviously decreased in the two groups , but TG decreased more obviously in the treatment group (P <0. 05). High density lipoprotein cholesterol (HDL-C) obviously increased in the treatment group (P <0. 05). Patient numbers of central obesity, uncontrolled hypertension, and uncontrolled diabetes obviously decreased and constituent numbers were obviously reduced in the treatment group after treatment, with better efficacy than those of the control group (P <0. 01 , P <0. 05). Conclusions YHR plus routine Western drugs could further reduce blood glucose, and had comprehensive interventional effects on multiple cardiovascular risk factors such as central obesity, blood lipids, and blood pressure in T2DM patients complicated with MetS. Its mechanism might be possibly correlated with improving insulin resistance and elevating insulin sensitivity of peripheral tissues.

[Effect of yiqi huaju recipe combined with routine therapy in treating hypertension patients with metabolic syndrome: a clinical study].

OBJECTIVE: To investigate the therapeutic effect of Yiqi Huaju Recipe (YHR) combined with routine therapy on the blood pressure, the blood pressure variability and other cardiovascular risk factors in hypertension patients complicated with metabolic syndrome (MetS). METHODS: Totally 43 hypertension patients complicated with MetS were recruited in this study and randomly assigned to the treatment group (22 cases, treated with basic routine treatment +YHR) and the control group (21 cases, treated with basic routine treatment + placebo). The treatment course was 12 weeks. Detected were parameters such as 24-h ambulatory blood pressure monitoring (ABPM), body mass index (BMI), waist circumference (WC), waist to hip ratio (WHR), homeostatic model assessment for insulin resistance (HOMA-IR), fasting glycosylated hemoglobin A1c (HbA1c), fasting plasma glucose (FPG), 2 h postprandial plasma glucose (2 h PPG), fasting plasma insulin (FPI), serum lipid, etc. RESULTS: The anthropometric parameters and plasma glucose levels (except HbAlc) were obviously lowered after treatment than before treatment in the treatment group (P < 0.01, P < 0.05). Besides, better effects were obtained in the WC, WHR, 2 h PPG, FPI and HOMA-IR (P < 0.05). The average blood pressure amplitude, the blood pressure variability, and blood pressure load at any time point were more obviously improved in the two groups after treatment than before treatment (P < 0.01, P < 0.05). Besides, partial indices were better in the treatment group than in the control group (P < 0.01, P < 0.05). CONCLUSIONS: YHR combined with routine therapy exhibited better effect on reducing the blood pressure amplitude, the blood pressure variability, and the blood pressure load in hypertension patients complicated with MetS. It could also effectively decrease the risk of other vascular disease.

Effect of a novel proteoglycan PTP1B inhibitor from Ganoderma lucidum on the amelioration of hyperglycaemia and dyslipidaemia in db/db mice.

Protein tyrosine phosphatase 1B (PTP1B) is implicated in the negative regulation of the insulin signalling pathway by dephosphorylating the insulin receptor (IR) and IR substrates. Ganoderma lucidum has traditionally been used for the treatment of diabetes in Chinese medicine; however, its anti-diabetic potency and mechanism in vivo is still unclear. Our previously published study reported a novel proteoglycan PTP1B inhibitor, named Fudan-Yueyang-Ganoderma lucidum (FYGL) from G. lucidum, with a half-maximal inhibitory concentration (IC50) value of 5·12 (sem 0·05) µg/ml, a protein:polyglycan ratio of 17:77 and 78 % glucose in polysaccharide, and dominant amino acid residues of aspartic acid, glycine, glutamic acid, alanine, serine and threonine in protein. FYGL is capable of decreasing plasma glucose in streptozotocin-induced diabetic mice with a high safety of median lethal dose (LD50) of 6 g/kg. In the present study, C57BL/6 db/db diabetic mice were trialed further using FYGL as well as metformin for comparison. Oral treatment with FYGL in db/db diabetic mice for 4 weeks significantly (P < 0·01 or 0·05) decreased the fasting plasma glucose level, serum insulin concentration and the homeostasis model assessment of insulin resistance. FYGL also controlled the biochemistry indices relative to type 2 diabetes-accompanied lipidaemic disorders. Pharmacology research suggests that FYGL decreases the plasma glucose level by the mechanism of inhibiting PTP1B expression and activity, consequently, regulating the tyrosine phosphorylation level of the IR ß-subunit and the level of hepatic glycogen, thus resulting in the improvement of insulin sensitivity. Therefore, FYGL is promising as an insulin sensitiser for the therapy of type 2 diabetes and accompanied dyslipidaemia.

MpAsr encodes an intrinsically unstructured protein and enhances osmotic tolerance in transgenic Arabidopsis.

Abscisic acid-, stress- and ripening (ASR) -induced proteins are plant-specific proteins whose expression is up-regulated under abiotic stresses or during fruit ripening. In this study, we characterized an ASR protein from plantain to explore its physiological roles under osmotic stress. The expression pattern of MpAsr gene shows that MpAsr gene changed little at the mRNA level, while the MpASR protein accumulates under osmotic treatment. Through bioinformatic-based predictions, circular dichroism spectrometry, and proteolysis and heat-stability assays, we determined that the MpASR protein is an intrinsically unstructured protein in solution. We demonstrated that the hydrophilic MpASR protein could protect L: -lactate dehydrogenase (L: -LDH) from cold-induced aggregation. Furthermore, heterologous expression of MpAsr in Escherichia coli and Arabidopsis enhanced the tolerance of transformants to osmotic stress. Transgenic 35S::MpAsr Arabidopsis seeds had a higher germination frequency than wild-type seeds under unfavorable conditions. At the physiological level, 35S::MpAsr Arabidopsis showed increased soluble sugars and decreased cell membrane damage under osmotic stress. Thus, our results suggest that the MpASR protein may act as an osmoprotectant and water-retaining molecule to help cell adjustment to water deficit caused by osmotic stress.

Inhibition of glycolysis by targeting lactate dehydrogenase A facilitates hyaluronan synthase 2 synthesis in synovial fibroblasts of temporomandibular joint osteoarthritis.

OBJECTIVE: Although associations between dysregulated glucose metabolism and human rheumatoid arthritis have been reported, the disturbance and influence of glycolytic metabolism on temporomandibular joint osteoarthritis remains unclear. This study aimed to investigate the expression level and metabolite profile of the critical glycolytic enzyme, lactate dehydrogenase A (LDHA) in synovial fibroblasts (SFs) of TMJOA, assess the effect of glycolytic inhibition on synthesis of hyaluronan synthase 2 (HAS2) and inflammation progression in these cells. METHODS: Immunohistochemistry and western blotting were performed to detect the expression of LDHA in the lining and sub-lining layers of synovial tissue and SFs. MTT and EdU assays were used to measure the cell proliferation. The cell apoptosis were demonstrated by TUNEL staining and Annexin V/PI double staining. A potent and specific inhibitor of LDHA, GSK2837808A, was administrated to suppress the activity of LDHA and detect the potential efficacy on HAS2. RESULTS: LDHA expression was dramatically higher in the synovial tissue and SFs from TMJOA patients compared to control groups. LDHA inhibition impaired active LDHA performance, suppressed the glucose uptake and decreased lactate concentration. Furthermore, GSK2837808A reversed the occurrence of low ratio of ATP/AMP, high level of Adenosine Monophosphate-activated Protein Kinase (AMPK) activation, disturbed HAS2 synthesis and hyaluronic acid (HA) production by inhibiting LDHA. The cellular viability and cell cycle were not affected by GSK2837808A at the working concentration. CONCLUSIONS: Targeting LDHA using its specific suppressant GSK2837808A impeded lactate secretion and contributed to HAS2 and HA synthesis in TMJOA SFs, providing the vital role of LDHA associated with TMJOA pathogenesis and a novel therapeutic approach for TMJOA.

Efficacy and Mechanism of a Chinese Classic Prescription of Yueju in Treating Nonalcoholic Steatohepatitis and Protecting Hepatocytes from Apoptosis.

Yueju, a famous classic Chinese prescription, has been extensively used in treating depression syndromes for hundreds of years. Recent studies have reported that Yueju showed good effects in treating metabolic diseases, such as obesity and hyperlipidemia. Nonalcoholic steatohepatitis (NASH), which leads to cirrhosis and severe cardiovascular diseases, is closely linked to obesity and abnormal lipid metabolism. In this study, Yueju could decrease the levels of alanine aminotransferase, aspartate transaminase, triglyceride, cholesterol, and low-density lipoprotein-C but increase the high-density lipoprotein-C in the serum of the NASH rat model induced by high-fat and high-cholesterol diet. Yueju could alleviate hepatosteatosis by increasing the phosphorylation of acetyl-CoA carboxylase and inhibiting the expression of fatty acid synthase and stearoyl-CoA desaturase 1. Yueju downregulated the expression of α-smooth muscle actin and collagen type 1A1, ameliorating the liver fibrilization. Yueju could also protect the hepatocytes from apoptosis by upregulating antiapoptosis protein Bcl-2 and X-linked inhibitor of apoptosis protein and downregulating apoptotic proteins Bax and cleaved poly ADP-ribose polymerase. Thus, Yueju could improve liver function, regulate lipid metabolism, alleviate hepatosteatosis and fibrosis, and protect hepatocytes from apoptosis against NASH. Yueju may be used as an alternative effective medicine for NASH treatment.

[Effects of berberine on cell proliferation, peroxisome proliferation activated receptor gamma, CAAT/enhancer binding protein mRNA and protein expression in 3T3-L1 pre-adipocytes].

OBJECTIVE: To study the effect and mechanism of berberine (BER) on the proliferation and differentiation of adipocytes. METHODS: The proliferation of 3T3-L1 pre-adipocytes was detected by XTT method. Lipid droplets accumulated in the cytoplasm of adipocytes in the differentiating process were observed by oil red O staining and quantified by colorimetry. The expressions of peroxisome proliferation activated receptor gamma (PPARgamma), CAAT/enhancer binding protein alpha (C/EBPalpha) mRNA and protein were detected by Real-time PCR and Western blotting respectively. RESULTS: Intervention with BER in concentration below 10 micromol/L for 24 h showed insignificant effect on the proliferation of adipocytes, as compared with that in the control group (P > 0.05); but that in concentrations 20, 40 and 80 micromol/L revealed significant suppressive effect; that in different concentrations acting for 48 h and 72 h could affect the proliferation and the effect displayed a dose-dependent manner, i. e. the higher the concentration of BER, the more apparent the suppression, showing significant difference as compared with those in the control group (P <0.05 or P <0.01). The pre-adipocyte treated with 10 micromol/L BER showed that the lipid droplets in the cytoplasm significantly lessened, so did the expression of differentiation related factor PPAR gamma mRNA as well as the expressions of C/EBPalpha mRNA and protein, as compared with those in the blank control group and the group intervened with rosiglitazone, the difference was significant (P < 0.05 or P < 0.01). CONCLUSIONS: BER can suppress the proliferation and differentiation of 3T3-L1 pre-adipocytes, reduce the accumulation of lipid drops in the adipocyte differentiating process, which may be associated with its effects in decreasing the expressions of adipocyte differentiation related gene PPARgamma, C/EBPalpha mRNA and protein. The study provides a basis for applying BER on the prevention and treatment of such metabolic related diseases as obesity.

[Effects of Pollen Typhae total flavones on mRNA expressions of peroxisome proliferator-activated receptors in 3T3-L1 adipocytes].

OBJECTIVE: To explore the effects of Pollen Typhae total flavones (PTF) on the mRNA expressions of peroxisome proliferator-activated receptors (PPARs) alpha,gamma, beta/delta so as to analyze its possible mechanism in improving the insulin sensitivity of 3T3-L1 adipocytes. METHODS: Adipocytes were treated with PTF, and expressions of PPARalpha, PPARgamma, PPARbeta/delta mRNAs relating to the adipocyte glucose and lipid metabolism were determined by reverse transcription polymerase chain reaction. RESULTS: PTF obviously up-regulated the expressions of PPARalpha and PPARgamma mRNAs, all showing significant differences as compared with those in the normal control group (P<0.01). CONCLUSION: With its function as an insulin sensitizer, PTF may enhance the PPARalpha and PPARgamma mRNA expressions in 3T3-L1 adipocytes.

[Pollen Typhae total flavones inhibit expression of interleukin-6 in C2C12 skeletal muscle cells cultured with palmitate].

OBJECTIVE: To observe the effects of Pollen Typhae total flavones (PTF) on expression of interleukin-6 (IL-6) mRNA and protein secretion in C2C12 cell strain of skeletal muscle cells cultured with palmitate, and to explore the mechanism of PTF in relieving insulin resistance (IR). METHODS: The IR of C2C12 cells was induced by co-culturing with palmitate. The C2C12 cells were divided into normal control group, untreated group, PDTC (a nuclear factor-kappaB inhibitor) treated group, rosiglitazone (ROS)-treated group, ROS+ PDTC-treated group, PTF-treated group and PTF+PDTC-treated group. Sixteen hours after culture, the transportation rate of glucose was observed by (3)H-deoxyglucose uptake method; IL-6 mRNA expression in C2C12 cells was assayed by real time polymerase chain reaction (Real-time PCR), and level of IL-6 protein secretion in culture supernatant was detected by enzyme linked immunosorbent assay. RESULTS: Compared with the normal control group, the transportation rate of glucose of cells in untreated group was decreased 30.43% after 16-hour palmitate culture, and was increased 32.39% in the PTF-treated group. Compared with the untreated group, the levels of IL-6 mRNA expression in cells and IL-6 protein secretion in supernatant were significantly decreased in the PTF-treated group (P<0.05). The levels of IL-6 mRNA expression in cells and IL-6 protein secretion in supernatant were increased in PTF+PDTC-treated group as compared with PFT-treated group (P<0.05). CONCLUSION: PTF can inhibit the IL-6 mRNA expression and IL-6 protein secretion via nuclear factor-kappaB pathway in C2C12 skeletal muscle cells, which may be one of its mechanisms in relieving inflammation conditions and insulin resistance in C2C12 skeletal muscle cells.

[Effects of Yiqi Sanju Formula on non-alcoholic fatty liver disease: a randomized controlled trial].

OBJECTIVE: To observe the therapeutic effects of Yiqi Sanju Formula (YQSJF), a compound traditional Chinese herbal medicine, in treatment of non-alcoholic fatty liver disease (NAFLD). METHODS: Sixty-seven patients diagnosed with NAFLD were randomly divided into two groups: YQSJF-treated group (39 cases) and placebo group (28 cases). The NAFLD patients in the two groups were treated with YQSJF and placebo respectively for 3 months. Clinical symptoms, the CT ratio of liver-spleen, body mass index (BMI), waist circumference, homeostatic model assessment for insulin resistance (HOMA2-IR) and the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), tumor necrosis factor-alpha (TNF-alpha), high sensitivity C-reactive protein (hs-CRP), triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL) and low-density lipoprotein (LDL) were evaluated before and after treatment. RESULTS: After treatment, the clinical symptoms were improved and the levels of BMI, waist circumference, HOMA2-IR, ALT, AST, TG and TC were decreased significantly in the YQSJF-treated group (P<0.05). The CT ratio of liver-spleen in the YQSJF-treated group was increased significantly as compared with the placebo group (P<0.01).

Protective effects of a G. lucidum proteoglycan on INS-1 cells against IAPP-induced apoptosis via attenuating endoplasmic reticulum stress and modulating CHOP/JNK pathways.

Fudan-Yueyang-G. lucidum (FYGL) is a water-soluble macromolecular proteoglycan extracted from Ganoderma lucidum which has been used for health promotion for a long time in China. The aim of this study was to investigate the protective effects of FYGL on INS-1 rat insulinoma beta cells against IAPP-induced cell apoptosis, as well as the underlying mechanisms. The results showed that apoptotic cells were significantly increased when incubated with islet amyloid polypeptide (IAPP). However, cytotoxicity of IAPP was significantly attenuated by co-incubation of the cells with FYGL. The results of RT-PCR showed that mRNA expression of caspase-3, caspase-12 and C/EBP homologous protein (CHOP) in IAPP-treated cells were inhibited by FYGL. Moreover, FYGL significantly prevented the IAPP-induced abnormal expression of inositol-requiring protein-1α (IRE1α), protein kinase RNA (PKR)-like ER kinase (PERK), activating transcription factor 6 (ATF6), as well as suppressed the activation of CHOP and c-Jun N-terminal kinase (JNK). Taken together, our results suggest that FYGL protects INS-1 pancreatic beta cells against IAPP-induced apoptosis through attenuating endoplasmic reticulum stress (ERS) and modulating CHOP/JNK pathways.

[Clinical research of Yiqi Sanju Formula in treating central obese men at high risk of metabolic syndrome].

OBJECTIVE: To observe the therapeutic effects of Yiqi Sanju Formula (YQSJF), a compound Chinese herbal medicine, on central obese men at high risk of metabolic syndrome (MS). METHODS: Compared with 30 healthy male volunteers, 45 central obese men were separated randomly into two groups and received the interventions with YQSJF and placebo respectively for 10 weeks. Baseline characteristics, insulin resistance, inflammation cytokines and plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (t-PA) were evaluated before and after treatment. RESULTS: The score of homeostatic model assessment for insulin resistance (HOMA-IR) and the levels of C reactive protein (CRP), free fatty acid (FFA) and PAI-1 in obese men were higher than those in the control group, while t-PA was lower. After treatment, compared with placebo group, body mass index, waist, and waist-to-hip ratio were decreased significantly in subjects who received YQSJF (P<0.01). The score of HOMA-IR and the levels of CRP, FFA and PAI-1 were decreased significantly, and the level of t-PA was increased significantly (P<0.01). CONCLUSION: YQSJF can reduce obesity and insulin resistance in central obese men at high risk of MS and improve inflammation and fibrinolysis, which indicates that it can reduce the risk of atherosclerosis.

Carrot antifreeze protein does not exhibit the polygalacturonase-inhibiting activity of PGIP family.

The carrot (Daucus carota) antifreeze protein (DcAFP) has a strong antifreeze activity and identified as belonging to the plant polygalacturonase-inhibiting protein (PGIP) family based on its sequence similarities, including the presence of a leucine-rich repeat (LRR) motif. In this study, yeast two-hybrid technology was used to analyze whether the carrot AFP could act as a PGIP. The complete DcAFP and polygalacturonase (PGase; obtained from fungus Alternaria alternata by RT-PCR) coding sequences were cloned into the bait and capture vectors, respectively, and yeast two-hybrid assays were performed. The results revealed that there was no evidence of an interaction between DcAFP and PGase, which suggests that DcAFP probably lacks PGIP activity. An analysis of the electrostatic potential of DcAFP and other PGIPs revealed that a large number of nonconservative residues within the beta-helix of the DcAFP LRR motif had been substituted to basic amino acids, thus changing the surface from negative to positive. This will electrostatically prevent DcAFP from binding with the positively charged surface of PGase. This is the first report that showed the correlation between nonconservative amino acids within the LRR motif of the DcAFP and its loss of polygalacturonase inhibiting activity.

[Effects of Pollen Typhae total flavone on glucose and lipid metabolism in 3T3-L1 adipocytes].

OBJECTIVE: To observe the effects of Pollen Typhae total flavone (PTF) on glucose and lipid metabolism in 3T3-L1 adipocytes. METHODS: The content of glucose which disappeared from the culture medium after incubation with drugs for 24 hours was determined as glucose consumption of the cells. The activity of cells was detected by XTT method. The transport of glucose was observed by (3)H-glucose uptake method. The efflux of free fatty acid (FFA) from adipocytes was observed by the concentration of FFA in the culture medium. RESULTS: The glucose concentration in culture medium was significantly decreased with a concentration-dependent effect, when PTF concentrations were from 0.025 g/L to 0.4 g/L. The toxic effect on cells appeared while PTF concentration was 0.4 g/L, and the MTT value decreased. PTF also significantly increased glucose transportation in the 3T3-L1 adipocytes as rosiglitazone (ROS) did. At the same time, FFA concentration in culture medium was significantly decreased as compared to the normal control group, while ROS-treated group did not show any difference. CONCLUSION: PTF can increase insulin sensitivity by increasing glucose transportation and consumption in the 3T3-L1 adipocytes as well as decreasing the FFA efflux from the cells.

Influence of Aluminium and EGCG on Fibrillation and Aggregation of Human Islet Amyloid Polypeptide.

The abnormal fibrillation of human islet amyloid polypeptide (hIAPP) has been implicated in the development of type II diabetes. Aluminum is known to trigger the structural transformation of many amyloid proteins and induce the formation of toxic aggregate species. The (-)-epigallocatechin gallate (EGCG) is considered capable of binding both metal ions and amyloid proteins with inhibitory effect on the fibrillation of amyloid proteins. However, the effect of Al(III)/EGCG complex on hIAPP fibrillation is unclear. In the present work, we sought to view insight into the structures and properties of Al(III) and EGCG complex by using spectroscopic experiments and quantum chemical calculations and also investigated the influence of Al(III) and EGCG on hIAPP fibrillation and aggregation as well as their combined interference on this process. Our studies demonstrated that Al(III) could promote fibrillation and aggregation of hIAPP, while EGCG could inhibit the fibrillation of hIAPP and lead to the formation of hIAPP amorphous aggregates instead of the ordered fibrils. Furthermore, we proved that the Al(III)/EGCG complex in molar ratio of 1 : 1 as Al(EGCG)(H2O)2 could inhibit the hIAPP fibrillation more effectively than EGCG alone. The results provide the invaluable reference for the new drug development to treat type II diabetes.

Effects of Pinus massoniana bark extract on cell proliferation and apoptosis of human hepatoma BEL-7402 cells.

AIM: To study the effects of Pinus massoniana bark extract (PMBE) on cell proliferation and apoptosis of human hepatoma BEL-7402 cells and to elucidate its molecular mechanism. METHODS: BEL-7402 cells were incubated with various concentrations (20-200 microg/mL) of PMBE for different periods of time. After 48 h, cell proliferation was determined by 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) assay. Apoptosis was evaluated by morphological observation, agarose gel electrophoresis, and flow cytometry analysis. Possible molecular mechanisms were primarily explored through immunohistochemical staining. RESULTS: PMBE (20-200 microg/mL) significantly suppressed BEL-7402 cell proliferation in a time- and dose-dependent manner. After treatment of BEL-7402 cells with 160 microg/mL PMBE for 24, 48, or 72 h, a typical apoptotic "DNA ladder" was observed using agarose gel electrophoresis. Nuclear condensation and boundary aggregation or split, apoptotic bodies were seen by fluorescence and electron microscopy. Sub-G1 curves were displayed by flow cytometry analysis. PMBE decreased the expression levels of Bcl-2 protein in a time-dependent manner after treatment of cells with 160 microg/mL PMBE. CONCLUSION: PMBE suppresses proliferation of BEL-7402 cells in a time- and dose-dependent manner and induces cell apoptosis by possibly downregulating the expression of the bcl-2 gene.

Significance of conservative asparagine residues in the thermal hysteresis activity of carrot antifreeze protein.

The approximately 24-amino-acid leucine-rich tandem repeat motif (PXXXXXLXXLXXLXLSXNXLXGXI) of carrot antifreeze protein comprises most of the processed protein and should contribute at least partly to the ice-binding site. Structural predictions using publicly available online sources indicated that the theoretical three-dimensional model of this plant protein includes a 10-loop beta-helix containing the approximately 24-amino-acid tandem repeat. This theoretical model indicated that conservative asparagine residues create putative ice-binding sites with surface complementarity to the 1010 prism plane of ice. We used site-specific mutagenesis to test the importance of these residues, and observed a distinct loss of thermal hysteresis activity when conservative asparagines were replaced with valine or glutamine, whereas a large increase in thermal hysteresis was observed when phenylalanine or threonine residues were replaced with asparagine, putatively resulting in the formation of an ice-binding site. These results confirmed that the ice-binding site of carrot antifreeze protein consists of conservative asparagine residues in each beta-loop. We also found that its thermal hysteresis activity is directly correlated with the length of its asparagine-rich binding site, and hence with the size of its ice-binding face.

Yiqi Huaju formula, a Chinese herbal medicine, reduces arterial pressure in salt­sensitive hypertension by inhibiting renin­angiotensin system activation.

Hypertension is a chronic disease with a high prevalence, and is associated with a high risk of vascular disease and premature death. Traditional Chinese medicine has been administered to treat hypertension for many years. In the present study, the effects of Yiqi Huaju formula (YQ; a compound used in traditional Chinese herbal medicine) were observed in salt­sensitive hypertension, which was induced by a high­salt and high­fat (HSF) diet and the potential mechanism was investigated. YQ was prepared from five plant extracts and was dissolved in normal sodium chloride prior to use. Male Sprague­Dawley rats were randomly divided into three groups, and fed either a normal diet (control), an HSF diet or an HSF diet with YQ. At week eight, blood pressure was measured and 24­h urine samples were collected from all of the rats. The rats were subsequently sacrificed, and their blood was collected for biochemical analyses and kidney tissue samples were dissected for the immunohistochemical assay. YQ was observed to decrease the high arterial pressure and serum total cholesterol level, which had been induced by the HSF diet. It also enhanced the excretion of urinary angiotensinogen, Na+, and decreased the loss of urinary aldosterone, K+ and microalbuminuria. In addition, YQ inhibited the high mRNA expression level of renal renin, angiotensin II (Ang II), and Ang II receptor, type 1 (AT1R), and inhibited the protein expression of renal AT1R and Ang II receptor type 2, which had been induced by the HSF diet. These results indicate that YQ may reduce the arterial pressure in salt­sensitive hypertension via the inhibition of renin­angiotensin system activation.

Marrow adiposity recovery after early zoledronic acid treatment of glucocorticoid-induced bone loss in rabbits assessed by magnetic resonance spectroscopy.

BACKGROUND: Although there is an inverse relationship between bone mass and marrow adiposity, the reversal function of zoledronic acid (ZOL) on increased marrow fat has not been studied. The aim of our study is to use the 3T magnetic resonance spectroscopy (MRS) to characterize the dynamical change process of the marrow fat responding to early ZOL treatment in the rabbit model with glucocorticoid-induced bone loss. METHODS: Fifteen 20-week-old female New Zealand White rabbits were randomized to control group, methylprednisolone (MPS) group, and MPS+ZOL group equally. Bone mineral density (BMD) and marrow fat fraction (FF) at L3-L4 vertebrae and left proximal femur were measured by Dual-energy X-ray absorptiometry and MRS at week 0, 4, 8, and 12. The animals were euthanized at the end of our experiment and their left femurs were dissected out for the histopathological examination. RESULTS: The MPS group demonstrated a remarkable increase in FF but a reduction in BMD compared with the controls at week 4 and 8, respectively (P<0.05 for all). Early treatment of ZOL can inhibit bone degeneration, although the bone mass would not recover to its original level. FF in MPS group exhibited a dramatic increase over time, with an increased FF variation (+31.6%, P=0.009) at week 4 from baseline and it was maintained until week 12 (+75.2%, P<0.001). In MPS+ZOL group, the FF returned to baseline value after the ZOL treatment. Comparing with the controls, larger marrow adipocyte density, the mean of the adipocyte diameter, and the percentage area of the adipocyte were observed in the MPS group (P<0.05 for all), whereas there were no significant differences in quantitative parameters of marrow adipocytes between the ZOL-treated group and the normal rabbits. CONCLUSION: An increase of the marrow adiposity is synchronized with the deterioration of the MPS-induced bone mass. A single dose of early ZOL can reverse the marrow adiposity to its original level completely.