Protective effect of aescin from the seeds of Aesculus hippocastanum on liver injury induced by endotoxin in mice

To investigate the effect and underlying mechanism of aescin on acute liver injury induced by endotoxin, liver injury was established by injecting lipopolysaccharide (LPS) in mice. Animals were assigned to seven groups: the control group and groups treated with LPS (40 mg/kg), aescin (3.6 mg/kg), LPS plus dexamethasone (4 mg/kg) and LPS plus aescin (0.9, 1.8 or 3.6 mg/kg). Hepatic histopathological changes were examined under a light microscope. Activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were determined. Levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), nitric oxide (NO) and antioxidative parameters in liver homogenate were measured. Glucocorticoid receptor (GR), 11 beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1) and 11 beta-hydroxysteroid dehydrogenase type 2 (11β-HSD2) expressions in liver were determined by western blotting. Treatment with escin could inhibit immigration of inflammatory cells, alleviate the degree of necrosis, and decrease serum ALT and AST activities. Aescin also down-regulated levels of inflammation mediators (TNF-α, IL-1β and NO) and 11β-HSD2 expression in liver, up-regulated GR expression, enhanced endogenous antioxidative capacity, but have no obvious effect on 11β-HSD1 expression in liver. The findings suggest aescin has protective effects on endotoxin-induced liver injury, and the underlying mechanisms were associated with its anti-inflammatory effects, up-regulating GR expression, down-regulating 11β-HSD2 experssion, and antixoidation.     

Association of monocyte chemoattractant protein-1 with adipocyte number, insulin resistance and liver function markers

Background  Monocyte chemoattractant protein-1 (MCP-1) is an inflammatory chemokine known to induce adipocyte dedifferentiation and insulin resistance. Inflammation, insulin resistance, and obesity have been implicated in the pathogenesis of non-alcoholic fatty liver disease (NAFLD).
Methods  Fasting plasma from 43 baboons were assayed for MCP-1, insulin, glucose, alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Adipocyte number and volume were measured via biopsies of omental adipose tissue. The homeostatic model assessment method (HOMA) was used to estimate systemic insulin resistance.
Results  Sex and age adjusted correlations were significant for MCP-1 with adipocyte number (r = −0.42; P  = 0.01), adipocyte volume (r = 0.38; P  = 0.02), HOMA (r = 0.45; P  = 0.004), ALT (r = 0.46; P  = 0.03) and AST (r = 0.45; P  = 0.03).
Conclusions  These results suggest that MCP-1 is related with adipocyte dedifferentiation and systemic insulin resistance, thereby potentially contributing to the development of NAFLD.     

Hepcidin protects against lipopolysaccharide-induced liver injury in a mouse model of obstructive jaundice

Obstructive jaundice (OJ) increases the risk of liver injury and sepsis, leading to increased mortality. Cholestatic liver injury is associated with a downregulation of hepcidin expression levels. In fact, hepcidin has an important antimicrobial effect, especially against Escherichia coli. It is unknown whether supplementing recombinant hepcidin is effective in alleviating cholestasis-induced liver injury and mortality in mice with superimposed sepsis. A mouse model of cholestasis was developed using extrahepatic bile duct ligation for 3 days. In addition, sepsis due to E. coli 0111:B4 lipopolysaccharide (LPS) was induced in the model. The serum levels of total bilirubin, AST, ALT, and LDH and the mRNA levels of IL-1β, TNF-α, and MCP-1 in the liver were significantly higher in the OJ mice receiving LPS than in the sham-operated mice receiving LPS. Compared to the OJ mice receiving LPS, the hepcidin-pretreated OJ mice receiving LPS showed a significant decrease in the above mentioned parameters, as well as a reversal in the downregulation of LC3B-II and upregulation of cleaved caspase-3; this, in turn, led to significantly decreased lethality in 24h. In conclusion, these results indicate that hepcidin pretreatment significantly reduced hepatic proinflammatory cytokine expression and liver injury, leading to reduced early lethality in OJ mice receiving LPS. Enhanced autophagy and reduced apoptosis may account for the protective effects of hepcidin.     

Effects of globin digest and its active ingredient Trp-Thr-Gln-Arg on galactosamine/lipopolysaccharide-induced liver injury in ICR mice

AimsWe investigated the effects of globin digest (GD) and its active ingredient Trp-Thr-Gln-Arg (WTQR) on galactosamine/lipopolysaccharide (GalN/LPS)-induced liver injury in imprinting control region (ICR) mice.Main methodsThe effects of WTQR and GD on the liver injury were examined by measuring the survival rate, serum aminotransferase activities, hepatic components, antioxidant enzyme activities, histopathological analysis, serum levels and hepatic gene expression of tumor necrosis factor-alpha (TNF-α), macrophage inflammatory protein-2 (MIP-2), and nitric oxide (NO) or inducible nitric oxide synthase (iNOS), and nuclear factor-kappa B (NF-κB) p65 content in GalN/LPS-treated ICR mice. RAW264 mouse macrophages were used to confirm the anti-inflammatory effects of WTQR and GD on the macrophages.Key findingsWTQR and GD increased the survival rate, suppressed the serum aminotransferase activities, serum levels and hepatic gene expression of TNF-α, MIP-2, and NO or iNOS, and nuclear NF-κB p65 content in GalN/LPS-treated mice; decreased the oxidized glutathione content, increased the superoxide dismutase activity, and decreased the histopathological grade values of the hepatocyte necrosis and lobular inflammation in GalN/LPS-injured liver; and suppressed the release levels and gene expression of TNF-α, MIP-2, and NO or iNOS, and nuclear NF-κB p65 content in LPS-stimulated RAW264 macrophages. WTQR and GD may improve the antioxidant defense system and inflammatory status in GalN/LPS-injured liver.SignificanceThese findings indicate that WTQR and GD have hepatoprotective effects on GalN/LPS-induced liver injury in ICR mice.     

Suramin prevents fulminant hepatic failure resulting in reduction of lethality through the suppression of NF-kappaB activity

AIM: Suramin is a symmetrical polysulfonated naphthylamine derivative of urea. There have been few studies on the effect of suramin on cytokines. We examined the effects of suramin on production of inflammatory cytokines. METHODS: We made an acute liver injury model treated with d-galactosamine (GalN) and lipopolysaccharide (LPS). Plasma AST, ALT, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-6 levels were measured. We compared with survival rate, histological found and NF-kappaB activity between with and without treatment of suramin. In macrophage like cell line, TNF-alpha and IL-6 production, TNF-alpha and IL-6 mRNA expression, and NF-kappaB activity was measured. RESULTS: The lethality of mice administered suramin with GalN/LPS was significantly decreased compared with that in mice without suramin. Changes of hepatic necrosis and apoptosis were slight in suramin-treated mice. Serum AST, ALT, TNF-alpha, IL-6 levels and NF-kappaB activity in the liver were significantly lower in mice administered suramin. In an in vitro model, suramin preincubation inhibited TNF-alpha and IL-6 production, TNF-alpha and IL-6 mRNA expression, and NF-kappaB activity. CONCLUSIONS: Suramin inhibits TNF-alpha and IL-6 production through the suppression of NF-kappaB activity from macrophages and shows therapeutic effects on acute liver damage.     

Protective effect of binaphthyl diselenide,a synthetic organoselenium compound,on 2‐nitropropane‐induced hepatotoxicity in rats

Organoselenides have been documented as promising pharmacological agents against a number of diseases associated with oxidative stress. Here we have investigated, for the first time, the potential antioxidant activity of binaphthyl diselenide ((NapSe)₂; 50 mg kg^?1, p.o.) against the 2‐nitropropane (2‐NP)‐induced hepatoxicity in rats, using different end points of toxicity (liver histopathology, plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT) and creatinine). In addition, in view of the association of oxidative stress with 2‐NP exposure, hepatic lipid peroxidation, ascorbic acid levels, δ‐aminolevulinate dehydratase (δ‐ALA‐D) and catalase (CAT) activities were evaluated. 2‐NP caused an increase of AST, ALT and hepatic lipid peroxidation. 2‐NP also caused hepatic histopathological alterations and δ‐ALA‐D inhibition. (NapSe)₂ (50 mg kg^?1) prevented 2‐NP‐induced changes in plasmatic ALT and AST activities and also prevented changes in hepatic histology, δ‐ALA‐D and lipid peroxidation. Results presented here indicate that the protective mechanism of (NapSe)₂ against 2‐NP hepatotoxicity is possibly linked to its antioxidant activity. Copyright © 2010 John Wiley & Sons, Ltd.     

柴芩承气汤对重症急性胰腺炎并发肝损伤大鼠的治疗作用及其机制

目的：研究柴芩承气汤（CQCQD）对重症急性胰腺炎（SAP）并发肝损伤大鼠的治疗作用及其机制。方法：72只SD大鼠随机分为3组（n=24）：假手术（sham）组,重症急性胰腺炎模型（SAP）组和柴芩承气汤治疗（CQCQD）组。去氧胆酸钠胰胆管逆行注射建立SAP大鼠模型,CQCQD组给予柴芩承气汤治疗,于造模后1 h、5 h、10 h观察各组不同时间点的胰腺、肝脏组织病理学变化,检测血清中淀粉酶（AMS）、丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）活性、白介素-6（IL-6）水平及胰腺、肝脏组织单核细胞趋化蛋白-1（MCP-1）和IL-6 mRNA的表达情况。结果：与sham组比较,SAP组血清AMS、ALT、AST活性及IL-6水平明显升高,胰腺、肝脏组织MCP-1及IL-6 mRNA表达升高（P<0.05）;与SAP组比较,CQCQD组血清AMS、ALT、AST活性及IL-6水平明显降低;胰腺和肝脏组织病理损伤减轻,胰腺、肝脏组织MCP-1及IL-6 mRNA表达明显减弱（P<0.05）。结论：MCP-1参与了SAP并发肝损伤的进展;柴芩承气汤能显著抑制胰腺、肝脏组织MCP-1的表达,减轻SAP时胰腺、肝脏组织病理损伤,对SAP并发肝损伤起到治疗作用。     

The relative efficacy of aminoguanidine and pentoxifylline in modulating endotoxin-induced cardiac stress

This study investigates the effect of aminoguanidine (AG), a selective inducible nitric oxide synthase (iNOS) inhibitor, and pentoxifylline (PTX), a tumour necrosis factor–alpha (TNF‐α) inhibitor, on lipopolysaccharide (LPS)‐induced cardiac stress. Rats were divided into four groups: group I served as a control, group II (LPS) received a single intraperitoneal injection of LPS (10 mg·kg^–1), group III (LPS+AG) and group IV (LPS+PTX) were injected with either AG (100 mg·kg^–1) or PTX (150 mg·kg^–1) intraperitoneally 10 days prior to LPS administration. Normalization of cardiac levels of nitrite/nitrate (NO_X), malondialdehyde (MDA), glutathione (GSH), heme oxygenase‐1 (HO‐1), glutathione peroxidase (GPx) and Na⁺, K⁺‐ATPase activities was evident in the AG group. Both AG and PTX decreased the elevated serum TNF‐α levels, the activities of lactate dehydrogenase (LDH), creatine kinase (CK) and cardiac myeloperoxidase (MPO). The levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and phosphocreatine (PCr) were enhanced following AG and PTX pretreatments. Calcium (Ca²⁺) levels were altered, and the histopathological observations supported the described results. Conclusively, the study highlights the cardioprotective potential of AG and PTX with superior results from AG. These findings reveal the relative contribution of nitric oxide and TNF‐α to oxidative stress and energy failure during endotoxemia. Copyright © 2011 John Wiley & Sons, Ltd.     

Bilirubin release induced by tumor necrosis factor in combination with galactosamine is toxic to mice

Application of tumor necrosis factor (TNF) in combination with galactosamine (GalN) in mice causes severe apoptosis of hepatocytes, resulting in complete destruction of the liver. Administration of high levels of unconjugated bilirubin and abnormally high production of unconjugated bilirubin have been reported to cause liver damage and are associated with several human pathologies. Serum alanine aminotransferase as well as total and direct bilirubin levels in mice were determined. Bilirubin levels are shown to significantly increase after a challenge with TNF/GalN in mice. Pretreatment with a heme oxygenase-1 inhibitor significantly prevents this release in bilirubin and offers significant protection against TNF/GalN-induced lethality. A correlation between the release of unconjugated bilirubin and the toxicity accompanied with this release is provided.     

Honokiol: an effective inhibitor of tumor necrosis factor-α-induced up-regulation of inflammatory cytokine and chemokine production in human synovial fibroblasts

In this study, we investigated the mechanisms underlying the anti-inflammatory effects of honokiol in tumor necrosis factor (TNF)-α-stimulated rheumatoid arthritis synovial fibroblasts (RASFs). RASFs pre-treated with honokiol (0-20 μM) were stimulated with TNF-α (20 ng/ml). The levels of prostaglandin E2 (PGE2), nitric oxide (NO), soluble intercellular adhesion molecule-1 (sICAM-1), transforming growth factor-β1 (TGF-β1), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1α (MIP-1α) in supernatants were determined by enzyme-linked immunosorbent assay (ELISA) and Griess assay. In addition, protein expression levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and phosphorylated Akt, nuclear factor kappa B (NFκB), and extracellular signal-regulated kinase (ERK)1/2 were determined by western blot. The expression of NFκB-p65 was assessed by immunocytochemical analysis. TNF-α treatment significantly up-regulated the levels of PGE2, NO, sICAM-1, TGF-β1, MCP-1, and MIP-1α in the supernatants of RASFs, increased the protein expression of COX-2, iNOS, and induced phosphorylation of Akt, IκB-α, NFκB, and ERK1/2 in RASFs. TNF-α-induced expression of these molecules was inhibited in a dose-dependent manner by pre-treatment with honokiol. The inhibitory effect of honokiol on NFκB-p65 activity was also confirmed by immunocytochemical analysis. In conclusion, honokiol is a potential inhibitor of TNF-α-induced expression of inflammatory factors in RASFs, which holds promise as a potential anti-inflammatory drug.     

Y-40138, a multiple cytokine production modulator, protects against D-galactosamine and lipopolysaccharide-induced hepatitis

Acute and fulminant liver failure induced by viral hepatitis, alcohol or other hepatotoxic drugs are associated with tumor necrosis factor (TNF) production. D-Galactosamine (D-GalN) and lipopolysaccharide (LPS)-induced liver injury is an experimental model of fulminant hepatic failure. In this model, TNF-alpha plays a central role in the pathogenesis of D-GalN/LPS-induced liver injury in mice. Y-40138, N-[1-(4-[4-(pyrimidin-2-yl)piperazin-1-yl]methyl phenyl)cyclopropyl] acetamide.HCl inhibits TNF-alpha and augments interleukin (IL)-10 production in LPS-injected mice in plasma. In the present study, we examined the effect of Y-40138 on D-GalN/LPS-induced hepatitis. Y-40138 (10mg/kg, i.v.) significantly suppressed TNF-alpha and monocyte chemoattractant protein-1 (MCP-1) production and augmented IL-10 production in plasma. In addition, Y-40138 significantly inhibited TNF-alpha production induced by direct interaction between human T lymphocytes and macrophages. Y-40138 suppressed plasma alanine transaminase (ALT) elevation and improved survival rate in D-GalN/LPS-injected mice, and it is suggested that the protective effect of Y-40138 on hepatitis may be mediated by inhibition of TNF-alpha and MCP-1, and/or augmentation of IL-10. This compound is expected to be a new candidate for treatment of cytokine and/or chemokine-related liver diseases such as alcoholic hepatitis.     

Differential regulation of C-C chemokines during fibroblast-monocyte interactions: adhesion vs. inflammatory cytokine pathways.

The cell-to-cell interactions during chronic inflammatory diseases likely contribute to leukocyte accumulation leading to increased pathology and organ dysfunction. In particular, there is a paucity of information relating to the maintenance of chronic fibrotic diseases. Using a lung fibroblast line and enriched monocyte populations, we have investigated the activational events which contribute to the production of two C-C chemokines, macrophage inflammatory protein-1 alpha (MIP-1alpha) and monocyte chemoattractant protein-1 (MCP-1), during fibroblast-monocyte interactions. Neither the fibroblast cell line (16lu) nor isolated monocytes alone produced significant levels of MIP-1alpha or MCP-1. However, when isolated monocytes were layered onto 16 lu fibroblast monolayers a significant increase in MIP-1alpha and MCP-1 production was observed. The use of fixed cell populations indicated that the MIP-1alpha was derived from monocytes and MCP-1 from both cell populations. To examine the molecules which were required for chemokine production during the interaction, specific antibodies were used in the co-cultures. Blocking beta3-integrin interactions significantly inhibited MIP-1alpha production. In contrast, beta-integrin interactions had no effect on the MCP-1 production, while, neutralization of TNF significantly decreased MCP-1 production during the co-culture. These data indicate that fibroblast-monocyte interactions induce chemokine production through different mechanisms and a combination of these responses may contribute to the maintenance of the mononuclear cell accumulation during disease progression.     

Preventive effect of omega-3 fatty acids in a rat model of stress-induced liver injury

Omega-3 fatty acids are gaining attention as a therapeutic agent of many diseases. Their protective effect in a variety of diseases has been demonstrated. To the best of our knowledge, this is the first study on omega-3 fatty acids related to acute cold-restraint stress (CRS) induced hepatic dysfunction in rats. Forty adult male Sprague–Dawley albino rats were used and classified into: control, omega-3 group, each rat was pretreated with omega-3 fatty acids; CRS group, rats were subjected to acute CRS for 6 hr; and CRS group pretreated with omega-3 fatty acids. Serum was obtained to determine corticosterone (CORT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and tumor necrosis factor-α (TNF-α) levels. Hepatic malondialdehyde (MDA) and total antioxidant capacity (TAC) levels were measured. Also, liver tissues were taken for histological examination and immunohistochemical assessment of the apoptotic marker, caspase-3. Results showed that pretreatment of stressed rats with omega-3 fatty acids led to significant decrease in hepatic MDA and increase in TAC levels. They reduced serum levels of CORT, ALT, AST, and TNF-α. Also, they improved liver damage and suppressed hepatic caspase-3 expression. In conclusion, pretreatment of stressed rats with omega-3 fatty acids has ameliorated stress-induced liver damage due to their antioxidant, anti-inflammatory, and antiapoptotic effects. So, they can be used to minimize stress complications on the liver.     

Role of mesenchymal stem cells on cornea wound healing induced by acute alkali burn

The aim of this study was to investigate the effects of subconjunctivally administered mesenchymal stem cells (MSCs) on corneal wound healing in the acute stage of an alkali burn. A corneal alkali burn model was generated by placing a piece of 3-mm diameter filter paper soaked in NaOH on the right eye of 48 Sprague-Dawley female rats. 24 rats were administered a subconjunctival injection of a suspension of 2×10(6) MSCs in 0.1 ml phosphate-buffered saline (PBS) on day 0 and day 3 after the corneal alkali burn. The other 24 rats were administered a subconjunctival injection of an equal amount of PBS as a control. Deficiencies of the corneal epithelium and the area of corneal neovascularization (CNV) were evaluated on days 3 and 7 after the corneal alkali burn. Infiltrated CD68(+) cells were detected by immunofluorescence staining. The mRNA expression levels of macrophage inflammatory protein-1 alpha (MIP-1α), tumor necrosis factor-alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1) and vascular endothelial growth factor (VEGF) were analyzed using real-time polymerase chain reaction (real-time PCR). In addition, VEGF protein levels were analyzed using an enzyme-linked immunosorbent assay (ELISA). MSCs significantly enhanced the recovery of the corneal epithelium and decreased the CNV area compared with the control group. On day 7, the quantity of infiltrated CD68(+) cells was significantly lower in the MSC group and the mRNA levels of MIP-1α, TNF-α, and VEGF and the protein levels of VEGF were also down-regulated. However, the expression of MCP-1 was not different between the two groups. Our results suggest that subconjunctival injection of MSCs significantly accelerates corneal wound healing, attenuates inflammation and reduces CNV in alkaline-burned corneas; these effects were found to be related to a reduction of infiltrated CD68(+) cells and the down-regulation of MIP-1α, TNF-α and VEGF.     

Hepatoprotective effects of IL-22 on fulminant hepatic failure induced by d-galactosamine and lipopolysaccharide in mice

Interleukin-22 (IL-22), a member of the IL-10 cytokine family that is produced by activated Th22, Th1 and Th17 cells as well as natural killer cells, plays an important role in increase of innate immunity, protection from damage and enhancement of regeneration. Here, we examined the effects of IL-22 on acute liver failure model induced by d-galactosamine (GalN) and lipopolysaccharide (LPS). Administration of recombinant human IL-22 (rhIL-22) reduced the death rate markedly and prevented mice from severe hepatic injury, as evidenced by decreased serum alanine aminotransferase (ALT) and total bilirubin (T.Bil) activity as well as improved histological signs in liver. Furthermore, IL-22 treatment decreased the hepatic malondialdehyde (MDA) contents and increased the reduced glutathione levels. Serum tumor necrosis factor α (TNF-α) level and hepatic caspase-3 activity were significantly lower in mice administrated with IL-22. Moreover, IL-22 treatment significantly enhanced activation of STAT3 and up-regulated the expression of Bcl-xL, heme oxygenase-1 (HO-1) and redox factor-1 (Ref-1) in the liver injury induced by GalN/LPS. Collectively, these data indicate that IL-22 can provide critical protection against GalN/LPS-induced liver injury through anti-apoptotic, anti-oxidant and anti-inflammatory actions.     

Inhibitory effect of curcumin on liver injury in a murine model of endotoxemic shock

The effect of curcumin on lipopolysaccharide/d-galactosamine (LPS/GalN)-induced acute shock model of liver injury was examined in mice. The simultaneous administration of LPS (5–20 μg kg⁻¹, i.p.) and GalN (700 mg kg⁻¹, i.p.) markedly increased the serum tumor necrosis factor-α (TNF-α), glutamic oxaloacetic transaminase/glutamic pyruvic transaminase (GOT/GPT), and massive hepatic necrosis and inflammation, leading to 100% lethality. Pre-administration of curcumin (100 mg kg⁻¹, i.p.) 3 h before induction with LPS/GalN imparted a large extent of protection against acute elevation in serum TNF-α and serum GOT/GPT. Hepatic necrosis and lethality caused by LPS/GalN was also greatly reduced by curcumin treatment. The results demonstrated that curcumin could protect mice from LPS/GalN-induced hepatic injury and inflammation through blockading TNF-α production, eventually raising the survival rate of septic-shock-induced mice.     

Serum levels of sRAGE, the soluble form of receptor for advanced glycation end products, are associated with inflammatory markers in patients with type 2 diabetes

Advanced glycation end products (AGEs) and their receptor (RAGE) play an important role in accelerated atherosclerosis in diabetes. We have recently found that the soluble form of RAGE (sRAGE) levels are significantly higher in type 2 diabetic patients than in nondiabetic subjects and positively associated with the presence of coronary artery disease in diabetes. In this study, we examined whether serum levels of sRAGE correlated with inflammatory biomarkers in patients with type 2 diabetes. Eighty-six Japanese type 2 diabetic patients (36 men and 50 women, mean age 68.4+/-9.6 years) underwent a complete history and physical examination, determination of blood chemistries, sRAGE, monocyte chemotactic protein-1 (MCP-1), adiponectin, tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6). Univariate regression analysis showed that serum levels of sRAGE positively correlated with alanine aminotransferase (ALT) (r=0.437, P=0.0001), MCP-1 (r=0.359, P=0.001), TNF-alpha (r=0.291, P=0.006), and hyperlipidemia medication (r=0.218, P=0.044). After multiple regression analyses, ALT (P<0.0001), MCP-1 (P=0.007), and TNF-alpha (P=0.023) remained significant. The present study demonstrates for the first time that serum levels of sRAGE are positively associated with MCP-1 and TNF-alpha levels in type 2 diabetic patients. These observations suggest the possibility that sRAGE level may become a novel biomarker of vascular inflammation in type 2 diabetic patients.