Baseline lymphocyte and cholinesterase levels may be the predictors of chronic herbal drug-induced liver injury.

Objective: To investigate the factors influencing the chronicity of drug-induced liver injury (DILI) caused by Chinese herbal medicine. Methods: Patients with DILI diagnosed by using the RUCAM score were enrolled retrospectively. The subjects were patients with DILI induced by taking Chinese herbal medicine and were followed up for 48 weeks. These patients were divided into a cure group and a chronic group. The biochemical indicators were monitored at baseline and every 3 months. Logistic regression was used to analyze the risk factors of DILI chronicity. The ROC (receiver operator characteristic) curve was used to analyze the diagnostic efficiency of each factor. Results: A total of 420 patients with DILI were enrolled; 122 of them were caused by Chinese herbal medicine, 70.5% (86/122) of them were female, chronic group 31.2% (39/122), and cure group 68.0% (83/122); cholinesterase (ChE) in the chronic group was lower than that in the cure group (5467.10 ± 2010.40 U/L vs. 6248.52 ± 1901.78 U/L, p = 0.04, t = 2.078). There was no significant difference in the age between cured patients and chronic patients (p = 0.156, Z = -1.417). There was no significant difference between the prognosis of different genders (p = 0.521, Z = -0.639). The logistic regression analysis showed that baseline lymphocyte (OR = 0.429, 95%CI = 0.205-0.898, p = 0.025) and cholinesterase (OR = 0.088, 95%CI = 0.008-0.994, p = 0.049) were independent risk factors of drug-induced chronicity. Conclusion: Baseline lymphocyte and cholinesterase may be the predictive factors for the chronicity of Chinese herbal medicine-induced liver injury.

Cytokine Profiles and Virological Marker Monitoring during 48 Weeks Peginterferon Alfa Treatment for HBeAg-Positive Chronic Hepatitis B.

Objective: This study aimed to investigate whether cytokine profiles and virological markers might add value in monitoring the effects of peginterferon (PEG-IFN) therapy for hepatitis B e-antigen (HBeAg) positive chronic hepatitis B (CHB). Methods: HBeAg positive patients with CHB were treated with PEG-IFN for 48 weeks. Clinical biochemical, and HBV serological indexes, as well as cytokines, were detected at baseline and every 12 weeks. Results: A total of 116 patients with CHB were enrolled in this study; 100 patients completed the 48-week treatment and follow-up, of whom 38 achieved serum HBeAg disappearance, 25 achieved HBeAg seroconversion, 37 showed HBsAg decreases ≥ 1 log 10 IU/mL, 9 showed HBsAg disappearance, and 8 became HBsAb positive. The cytokine levels at baseline and during treatment were similar between the HBeAg disappearance group and non-disappearance group. The disappearance of HBeAg was independently associated with HBeAg levels at weeks 12 and 24, and with the HBeAg decline at week 24 ( P < 0.05). The HBsAg response was independently associated with HBsAg, the HBsAg decline, HBeAg, the HBeAg decline at week 12, and HBsAg at week 24 ( P< 0.05). Conclusion: There was no significant correlation between the response to interferon (IFN) and cytokines during PEG-IFN treatment. The changes in virological markers predicted the response to IFN after 48 weeks.

Combination and Intermittent Therapy Based on Pegylated Interferon Alfa-2a for Chronic Hepatitis B with Nucleoside (Nucleotide) Analog-Experienced Resulting in Hepatitis B Surface Antigen Clearance: A Case Report.

Interferon (IFN) and nucleoside (nucleotide) analogs (NAs) are two effective antiviral drugs for chronic hepatitis B (CHB). More and more evidence shows that the combination of the two drugs can better inhibit viral replication and even achieve clinical cure. IFN intermittent therapy is also considered to be an important measure to resolve IFN fatigue when hepatitis B surface antigen (HBsAg) decline appears stagnated during IFN-based antiviral therapy. A 36-year-old male NA-experienced patient with hepatitis B e antigen (HBeAg)-positive CHB was admitted to our hospital. After a poor response to tenofovir disoproxil fumarate (TDF) monotherapy for 1 year, the patient was treated with pegylated interferon alfa-2a combination therapy and finally achieved HBsAg clearance. During the treatment and follow-up, HBsAg, HBeAg, hepatitis B virus (HBV) DNA, and serum alanine aminotransferase, etc. were monitored every 3 months. Between weeks 58 and 71 of combination therapy, IFN was discontinued because of a slow decline in HBsAg, and TDF alone was used for maintenance therapy. Complete virological response, HBeAg and HBsAg seroconversion were observed at weeks 44, 96, and 122, respectively. After 24 weeks of consolidation therapy, HBsAg, HBeAg, and HBV DNA were consistently negative, and hepatitis B surface antibody was 729.30 mIU/mL at week 146 of the combination therapy, then we stopped drugs. Following up after 28 weeks of cessation therapy, the patient still remained clinically cured.

Changes in the Cytokine Profiles of Patients with Chronic Hepatitis B during Antiviral Therapy.

OBJECTIVE: To investigate the changes in the cytokine profiles of chronic hepatitis B (CHB) patients undergoing antiviral treatment. METHODS: Hepatitis B e antigen (HBeAg)-positive patients were treated with Pegylated interferon (PEG-IFN) and entecavir (ETV). Clinical biochemistry and cytokines were detected at baseline and every 3 months. RESULTS: In all, 200 patients completed 48 weeks of treatment, 100 in the PEG-IFN group and 100 in the ETV group. During 3-6 months of treatment, compared with baseline, the PEG-IFN group showed a significant decrease in interferon-gamma (IFN-γ), interleukin-17A (IL-17A), interleukin-6(IL-6), interleukin-10(IL-10), and transforming growth factor beta (TGF-ß) ( P < 0.001) and a significant increase in interferon-alpha 2(IFN-α2) ( P < 0.001). In the ETV group, IL-10 and TGF-ß1 decreased significantly ( P < 0.001). After 3 months, the levels of IFN-α2, IL-17A, and tumor necrosis factor-alpha(TNF-α) in the PEG-IFN group were significantly higher than those in the ETV group ( P < 0.01). The levels of IL-6 and TGF-ß3 were significantly lower than those in the ETV group ( P < 0.01). After 6 months, the levels of IFN-α2, IFN-γ, and TNF-α in the PEG-IFN group were significantly higher than those in the ETV group ( P < 0.01), while the levels of IL-6 and TGF-ß3 were significantly lower than those in the ETV group ( P < 0.01). Compared with ETV, PEG-IFN had higher HBeAg and HBsAg disappearance rates. CONCLUSION: During antiviral therapy, a change in the cytokine profile occurred; in the aspect of immune control and functional cure, PEG-IFN was significantly better than ETV.

Using proteomics to discover novel biomarkers for fatty liver development and response to CB1R antagonist treatment in an obese mouse model.

Over activity of cannabinoid receptor type 1 (CB1R) plays a key role in increasing the incidence of obesity-induced non-alcoholic fatty liver disease. Tissue proteome analysis has been applied to investigate the bioinformatics regarding the mode of action and therapeutic mechanism. The aim of this study was to explore the potential pathways altered with CB1R in obesity-induced fatty liver. Male C57BL/6 mice were fed either a standard chow diet (STD) or a high-fat diet (HFD) with or without 1-week treatment of CB1R inverse agonist AM251 at 5 mg/kg. Then, liver tissues were harvested for 2DE analysis and protein profiles were identified by using MALDI-MS. Results showed that eight of significantly altered protein spots at the level of changes > twofold were overlapped among the three groups, naming major urinary protein 1, ATP synthase subunit ß, glucosamine-fructose-6-phosphate aminotransferase 1, zine finger protein 2, s-adenosylmethionine synthase isoform type-1, isocitrate dehydrogenase subunit α, epoxide hydrolase 2 and 60S acidic ribosomal protein P0. These identified proteins were involved in glucose/lipid metabolic process, xenobiotic metabolic system, and ATP synthesized process in mitochondria. Based on the findings, we speculated that CB1R blockade might exert its anti-metabolic disorder effect via improvement of mitochondrial function in hepatic steatosis in HFD condition.

Cannabinoid receptor type 1 mediates high-fat diet-induced insulin resistance by increasing forkhead box O1 activity in a mouse model of obesity.

Hepatic glucose production is promoted by forkhead box O1 (FoxO1) under conditions of insulin resistance. The overactivity of cannabinoid receptor type 1 (CB1R) partly causes increased liver fat deposits and metabolic dysfunction in obese rodents by decreasing mitochondrial function. The aim of the present study was to investigate the role of FoxO1 in CB1R-mediated insulin resistance through the dysregulation of mitochondrial function in the livers of mice with high-fat diet (HFD)-induced obesity. For this purpose, male C57BL/6 mice were randomly assigned to groups and either fed a standard diet (STD), a HFD, or a HFD with 1-week treatment of the CB1R inverse agonist, AM251, at 1 or 5 mg/kg. For in vitro experiments, AML12 hepatocytes were incubated with FoxO1 siRNA prior to challenge with arachidonyl-2'-chloroethylamide (ACEA) or a high concentration of free fatty acids (HFFA). Plasma parameters were analyzed using colorimetric methods. Liver histopathology and hepatic status markers were examined. The HFD-fed mice exhibited an increase in CB1R levels in the liver. Moreover, in response to increased hepatic oxidative stress, the HFD-fed mice also displayed hepatic mitochondrial dysfunction, as indicated by the decreased mRNA levels of carnitine palmitoyltransferase-1 (CPT-1), mitochondrial transcription factor A (TFAM), nuclear respiratory factor-1 (NRF-1) and citrate synthase. On the contrary, these effects in the HFD-fed mice were reversed by treatment with 5 mg/kg AM251. The administration of AM251 suppressed the induction of FoxO1, phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase) expression in the livers of the mice fed a HFD by enhancing the phosphorylation of insulin signaling cascades thus, further lowering the high level of the homeostatic model assessment of insulin resistance (HOMA­IR) index. In our in vitro experiments, transfection with FoxO1 siRNA prevented the HFFA- and ACEA-induced decrease in the gene expression of mitochondrial biogenesis-related factors, and abrogated the HFFA- and ACEA-induced increase in PEPCK and G6Pase expression. Taken together, our findings suggest that the anti-insulin resistance effect of AM251, which leads to an improvement of mitochondrial function in hepatic steatosis, is mediated through FoxO1.

Major urinary protein 1 interacts with cannabinoid receptor type 1 in fatty acid-induced hepatic insulin resistance in a mouse hepatocyte model.

Hepatic insulin resistance (HIR) is a metabolic abnormality characterized by increased gluconeogenesis which usually contributes from an elevation of free fatty acids. Cannabinoid receptor type 1 (CB1R) and major urinary protein 1 (MUP1) are thought to play pivotal roles in mitochondrial dysfunction, liver steatosis and insulin resistance. The aim of this study was to explore the role of MUP1 in CB1R-mediated HIR through the dysregulation of mitochondrial function in AML12 mouse hepatocytes challenged with high concentration of free fatty acids (HFFA). Firstly we observed that treatment of AM251, a selective CB1R antagonist, obviously reversed the HFFA-induced reduction of MUP1 protein expression both in vivo and in vitro. Additionally, our results revealed that AM251 also reverted HFFA-mediated decrease of the mRNA level of mitochondrial biogenesis-related factors, mtDNA amount, ATP production, mitochondrial respiratory complexes-I and -III, and mitochondrial membrane potential, thus consequently might correlate with a parallel reduction of ROS production. Meanwhile, AM251 attenuated HFFA-induced impairment of insulin signaling phosphorylation and elevation of phosphoenolpyrvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase), two key enzymes of gluconeogenesis. Silence of MUP1 gene abolished the inhibitory effect of AM251 on HFFA-mediated elevation of PEPCK and G6Pase expression, whereas the suppression of insulin signaling and mRNA level of mitochondrial biogenesis-related factors were only partially recovered. Altogether, these findings suggest that the anti-HIR effect of AM251 via improvement of mitochondrial functions might occur in a MUP1-dependent manner.

Endothelin-1 exacerbates development of hypertension and atherosclerosis in modest insulin resistant syndrome.

Endothelin-1 (ET-1) is known as potent vasoconstrictor, by virtue of its mitogenic effects, and may deteriorate the process of hypertension and atherosclerosis by aggravating hyperplasia and migration in VSMCs. Our previous study demonstrated that insulin infusion caused sequential induction of hyperinsulinemia, hyperendothelinemia, insulin resistance, and then hypertension in rats. However, the underlying mechanism of ET-1 interfere insulin signaling in VSMCs remains unclear. To characterize insulin signaling during modest insulin resistant syndrome, we established and monitored rats by feeding high fructose-diet (HFD) until high blood pressure and modest insulin resistance occurred. To explore the role of ET-1/ETAR during insulin resistance, ETAR expression, ET-1 binding, and insulin signaling were investigated in the HFD-fed rats and cultured A-10 VSMCs. Results showed that high blood pressure, tunica medial wall thickening, plasma ET-1 and insulin, and accompanied with modest insulin resistance without overweight and hyperglycemia occurred in early-stage HFD-fed rats. In the endothelium-denuded aorta from HFD-fed rats, ETAR expression, but not ETBR, and ET-1 binding in aorta were increased. Moreover, decreasing of insulin-induced Akt phosphorylation and increasing of insulin-induced ERK phosphorylation were observed in aorta during modest insulin resistance. Interestingly, in ET-1 pretreated VSMCs, the increment of insulin-induced Akt phosphorylation was decreased whereas the increment of insulin-induced ERK phosphorylation was increased. In addition, insulin potentiated ET-1-induced VSMCs migration and proliferation due to increasing ET-1 binding. ETAR antagonist reversed effects of ET-1 on insulin-induced signaling and VSMCs migration and proliferation. In summary, modest insulin resistance syndrome accompanied with hyperinsulinemia leading to the potentiation on ET-1-induced actions in aortic VSMCs. ET-1 via ETAR pathway suppressed insulin-induced AKT activation, whereas remained insulin-induced ERK activation. ET-1 and insulin synergistically potentiated migration and proliferation mainly through ETAR/ERK dependent pathway, which is dominant in VSMCs during modest insulin resistance syndrome. Therefore, ET-1 and ETAR are potential targets responsible for the observed synergism effect in the hypertensive atherosclerotic process through enhancement of ET-1 binding, ET-1 binding, ETAR expression, and ET-1-induced mitogenic actions in aortic VSMCs.

Angiotensin II enhances endothelin-1-induced vasoconstriction through upregulating endothelin type A receptor.

Endothelin-1 (ET-1) is the most potent vasoconstrictor by binding to endothelin receptors (ETAR) in vascular smooth muscle cells (VSMCs). The complex of angiotensin II (Ang II) and Ang II type one receptor (AT1R) acts as a transient constrictor of VSMCs. The synergistic effect of ET-1 and Ang II on blood pressure has been observed in rats; however, the underlying mechanism remains unclear. We hypothesize that Ang II leads to enhancing ET-1-mediated vasoconstriction through the activation of endothelin receptor in VSMCs. The ET-1-induced vasoconstriction, ET-1 binding, and endothelin receptor expression were explored in the isolated endothelium-denuded aortae and A-10 VSMCs. Ang II pretreatment enhanced ET-1-induced vasoconstriction and ET-1 binding to the aorta. Ang II enhanced ETAR expression, but not ETBR, in aorta and increased ET-1 binding, mainly to ETAR in A-10 VSMCs. Moreover, Ang II-enhanced ETAR expression was blunted and ET-1 binding was reduced by AT1R antagonism or by inhibitors of PKC or ERK individually. In conclusion, Ang II enhances ET-1-induced vasoconstriction by upregulating ETAR expression and ET-1/ETAR binding, which may be because of the AngII/Ang II receptor pathways and the activation of PKC or ERK. These findings suggest the synergistic effect of Ang II and ET-1 on the pathogenic development of hypertension.

Endothelin-1 exacerbates lipid accumulation by increasing the protein degradation of the ATP-binding cassette transporter G1 in macrophages.

Endothelin-1 (ET-1), a potent proatherogenic vasoconstrictive peptide, is known to promote macrophage foam cell formation via mechanisms that are not fully understood. Excessive lipid accumulation in macrophages is a major hallmark during the early stages of atherosclerotic lesions. Cholesterol homeostasis is tightly regulated by scavenger receptors (SRs) and ATP-binding cassette (ABC) transporters during the transformation of macrophage foam cells. The aim of this study was to investigate the possible mechanisms by which ET-1 affects lipid accumulation in macrophages. Our results demonstrate that oxidized low-density lipoprotein (oxLDL) treatment increases lipid accumulation in rat bone marrow-derived macrophages. Combined treatment with ET-1 and oxLDL significantly exacerbated lipid accumulation in macrophages as compared to treatment with oxLDL alone. The results of Western blotting show that ET-1 markedly decreased the ABCG1 levels via ET type A and B receptors and activation of the phosphatidylinositol 3-kinase pathway; however, ET-1 had no effect on the protein expression of CD36, SR-BI, SR-A, or ABCA1. In addition, real-time PCR analysis showed that ET-1 treatment did not affect ABCG1 mRNA expression. We also found that ET-1 decreases ABCG1 possibly due to the enhancement of the proteosome/calpain pathway-dependent degradation of ABCG1. Moreover, ET-1 significantly reduced the efficiency of the cholesterol efflux in macrophages. Taken together, these findings suggest that ET-1 may impair cholesterol efflux and further exacerbate lipid accumulation during the transformation of macrophage foam cells.