A molecular and computational study of galbanic acid as a regulator of Sirtuin1 pathway in inhibiting lipid accumulation in HepG2 cells.

INTRODUCTION: Sirtuin1 (SIRT1) plays a crucial role in the pathophysiology of non-alcoholic fatty liver disease. We investigated the mechanistic role of galbanic acid (Gal) as a regulator of SIRT1 in silico and in vitro. METHODS: HepG2 cells were treated with Gal in the presence or absence of EX-527, a SIRT1-specific inhibitor, for 24 h. Sirtuin1 gene and protein expression were measured by RT-PCR and Western blotting, respectively. It has been docked to the allosteric reign of SIRT1 (PDB ID: 4ZZJ) to study the effect of Gal on SIRT1, and then the protein and complex molecular dynamic (MD) simulations had been studied in 100 ns. RESULTS: The semi-quantitative results of Oil red (p < .03) and TG level (p < .009) showed a significant reduction in lipid accumulation by treatment with Gal. Also, a significant increase was observed in the gene and protein expression of SIRT1 (p < .05). MD studies have shown that the average root mean square deviation (RMSD) was about 0.51 Å for protein structure and 0.66 Å for the complex. The average of radius of gyration (Rg) is 2.33 and 2.32 Å for protein and complex, respectively, and the pattern of root mean square fluctuation (RMSF) was almost similar. CONCLUSION: Computational studies show that Gal can be a great candidate to use as a SIRT1 ligand because it does not interfere with the structure of the protein, and other experimental studies showed that Gal treatment with SIRT1 inhibitor increases fat accumulation in HepG2 cells.

A pilot study evaluating high dose esomeprazole, bismuth subcitrate and amoxicillin for eradicating Helicobacter pylori infection in Iran.

BACKGROUND: Helicobacter pylori (H. pylori) is strongly associated with peptic ulcer disease and gastric cancer. We evaluated two triple therapy regimens comprising esomeprazole, high dose bismuth, and different doses of amoxicillin for first-line H. pylori eradication. MATERIALS AND METHODS: Two hundred patients with dyspepsia and naive H. pylori infection were randomly assigned into two groups (n = 100). Both groups were treated for 14 days similarly with esomeprazole (40 mg, twice daily) and bismuth subcitrate (240 mg, three times daily), but the dose of amoxicillin was varied between Groups A (750 mg) and B (1000 mg) three times daily. Treatment compliance and side effect were evaluated following the therapies and after 8 weeks, a negative test of stool H. pylori antigen confirmed eradication. RESULTS: The two groups were comparable with respect to sex and age. According to intention to treat analysis, eradication rates were 80% (95% CI: 77.2%-82.8%) and 90% (95% CI: 84.1%-95.9%) in A and B groups, respectively (p = 0.22). Per-protocol eradication rates were 87% (95% CI: 80.4%-93.6%) and 92.8% (95% CI: 87.7%-97.9%), respectively (p = 0.23). Severe adverse effects were 3% and 2%, respectively (p = 0.34). CONCLUSION: High dose esomeprazole, amoxicillin and bismuth achieved 92.8% cure rates per protocol in a country with a high background rate of resistance. Additional studies are needed to ascertain whether this therapy can be further improved. Until then, it can be recommended as a first-line H. pylori eradication in north of Iran.

Non-inferiority of reverse hybrid regimen versus standard concomitant regimen for H. pylori eradication in a randomized-controlled trial.

Background: Helicobacter pylori (H. pylori) infection is strongly related to peptic ulcer disease, chronic gastritis, and gastric malignancies. Therefore, H. pylori eradication is necessary in these cases. This study was aimed to compare the efficacy of 14-day reverse hybrid therapy with standard 14-day concomitant regimen for H. pylori eradication in Iran. Methods: Of the 317 patients with dyspepsia and H. pylori infection enrolled in the study, 153 and 164 patients were randomly assigned to reverse hybrid and concomitant groups, respectively. The reverse hybrid regimen containing pantoprazole, amoxicillin, clarithromycin, and metronidazole was taken every 12 hours in the first 7 days, however, Clarithromycin and Metronidazole were discontinued within the next 7 days. Patients in the concomitant group also received the same drugs for 14-day. Eradication confirmation tests were used 8 weeks after the end of treatments. Results: A crowd of 281 patients continued the trial until the end. H. pylori eradication rates based on intention to treat analysis were 71.2% (109/153) and 83.5% (137/164) in reverse hybrid and concomitant groups, respectively (P = 0.007). By the per-protocol analysis, rates of eradication were 85.8% (109/127) and 89% (137/154), respectively (P = 0.428). Severe side effects were few in both groups. More side effects were observed in concomitant group (p < 0.001), however, the severity of side effects was not statistically different between the two regimens (P = 0.314). Reverse hybrid regimen was better tolerated (98% vs. 91.5%, P = 0.009). Conclusion: Both 14-day reverse hybrid and concomitant regimens have a fair response rate in Iran.

Serum calprotectin can indicate current and future severity of COVID-19.

BACKGROUND: Predictive and prognostic biomarkers to guide 2019 novel coronavirus disease (COVID-19) are critically evolving. Dysregulated immune responses are the pivotal cause of severity mainly mediated by neutrophil activation. Thus, we evaluated the association of calprotectin, neutrophil secretory protein, and other mediators of inflammation with the severity and outcomes of COVID-19. METHODS: This two-center prospective study focused on PCR-proven COVID-19 patients (n = 76) with different clinical presentations and SARS-CoV-2 negative control subjects (n = 24). Serum calprotectin (SC) was compared with IL-6 and other laboratory parameters. RESULTS: Median levels of SC were significantly higher in COVID-19 patients in comparison to the control group (3760 vs. 2100 ng/ml, p < 0.0001). Elevated SC was significantly respective of disease severity (3760 ng/ml in mild up to 5700 ng/ml in severe cases, p < 0.0001). Moreover, the significant positive and negative correlations of SC with disease severity and oxygenation status indicated disease progression and respiratory worsening, respectively. It was found that SC was high in severe patients during hospitalization and significantly declined to normal after recovery. The logistic analysis identified the independent predictive power of SC for respiratory status or clinical severity. Indeed, SC behaved as a better discriminator for both outcomes, as it exhibited the largest area under the curve (receiver operating curve analysis), with the highest specificity and sensitivity when the predictive value of inflammatory biomarkers was compared. CONCLUSION: Calprotectin can be used as a reliable prognostic tool to predict the poor clinical outcomes of COVID-19 patients.

Efficacy of 14-day concomitant quadruple therapy and 14-day high-dose dual therapy on H. pylori eradication.

Aim: We compared the efficacy of two different regimens for H. pylori eradication in areas with high antibiotic resistance. Background: Helicobacter pylori (H. pylori) is a gram-negative bacillus that has a strong association with chronic gastritis and peptic ulcer disease. Different regimens with varying degrees of effectiveness have been used for H. pylori eradication. Methods: The current randomized controlled trial (RCT) randomly assigned 217 patients who had indications for H. pylori eradication therapy to two groups. One group were administered concomitant quadruple therapy (pantoprazole 40 mg, amoxicillin 1 gr, clarithromycin 500 mg, and metronidazole 500 mg every 12 hours) for 14 days, and the second group received 14 days of high-dose dual therapy, consisting of esomeprazole 40 mg BID and amoxicillin 1g TDS. H. pylori eradication was assessed eight weeks after the end of treatment. Results: H. pylori eradication rates by PP analysis for 14 days concomitant quadruple therapy and high-dose dual therapy were 88.6% (95% CI, 80.3-92.8) and 82.2% (95% CI, 74.8-89.5), respectively (p = 0.19). According to intention-to-treat (ITT) analysis, the eradication rates were 81.6% (95% CI, 74.5-88.6) and 80.6% (95% CI, 73-88.1), respectively (p = 0.58). Overall drug side effects were 20.8% in high-dose dual therapy and 49.6% in concomitant quadruple therapy (p < 0.001). Conclusion: Fourteen days concomitant quadruple therapy can be considered as a relatively acceptable regimen for H. pylori eradication in areas with high clarithromycin and metronidazole resistance. It seems that high-dose dual therapy could be a promising alternative regimen in these areas.

Chlorogenic acid improves anti-lipogenic activity of metformin by positive regulating of AMPK signaling in HepG2 cells.

Metformin improves lipid profile, however, combination therapy is developing to increase its effectiveness and reduce the deleterious effects of metformin. Chlorogenic acid (CGA) has exhibited lipid-lowering effects. This study aimed to investigate the combined effect of metformin and CGA on lipid accumulation, as well as to elucidate the engaged mechanism in HepG2 cells. To find the non-lethal doses of metformin and CGA, MTT assay was performed. High Glucose (HG) at 33 mM was used to induce lipogenesis in HepG2 cells. Following treatment with different concentrations of metformin and CGA, total lipid content (Oil Red O-staining), triglyceride level, the genes expression of SREBP-1c and FAS, and phosphorylation of AMPK and ACC were measured. Both Metformin and CGA decreased HG-induced lipid accumulation individually, by decreasing total lipid content and triglyceride level. The lowest effective doses of metformin and CGA were 0.25 mM and 5 µM, respectively, which significantly reduced SREBP-1c and FAS genes expression. The combination of these concentrations reinforced these effects. The phosphorylation of AMPK and ACC were more increased by metformin in combination with CGA than both individually. Our findings suggest that CGA synergistically enhances metformin lipid reducing action via the regulating of involved factors in fatty acid synthesis. Therefore, co-administration of metformin with CGA may have further medical value in treating lipid metabolism disorders.

Aberrant methylation of miR-124 upregulates DNMT3B in colorectal cancer to accelerate invasion and migration.

The dysregulation of microRNA expression is significantly associated with the initiation and development of CRC. miR-124 is markedly downregulated in colorectal cancer. In the present study, the effects of methylation, over expression and downregulation of miR-124 and its target gene DNMT3B on the proliferation, migration and invasion of colorectal cell line were investigated. The promoter methylation status of miR-124 in the CRC was investigated by methylation specific PCR (MSP). The potential role of miR-124 expression in CRC cells was investigated using the demethylation reagent 5-Aza-CdR and transfection of miR-124 mimic/antimir. MSP revealed that miR-124 promoter region was hypermethylated, result in its significant downregulation in tumour tissues. We showed miR-124 expression was upregulated following 5-AZA-CdR treatment. Transfected Hct-116 cell line with miR-124 leads to decreased DNMT3B expression, cell proliferation, migration and invasion of HCT-116. In conclusion, our data indicate that miR-124 suppress colorectal cancer proliferation, migration and invasion through downregulating DNMT3B level.

Elevated fecal and serum calprotectin in COVID-19 are not consistent with gastrointestinal symptoms.

Intestinal epithelial cell damage caused by SARS-CoV-2 infection was thought to be associated with gastrointestinal symptoms and decreased fecal consistency. The association of the gastrointestinal symptoms with the COVID-19-mediated inflammatory response triggered by the gastrointestinal immune system was investigated in this paper. Intestinal inflammation marker fecal calprotectin along with serum calprotectin and other inflammatory markers were measured in COVID-19 cases with and without GI manifestations as well as healthy individuals. Analyses were performed to compare COVID-19 patient subgroups and healthy controls and examine the relationship between fecal and serum calprotectin levels with gastrointestinal symptoms and disease severity. COVID-19 patients (n = 70) were found to have markedly elevated median levels of fecal (124.3 vs. 25.0 µg/g; P < 0/0001) and serum calprotectin (3500 vs. 1060 ng/mL; P < 0/0001) compared with uninfected controls. Fecal and serum calprotectin levels were not significantly different between COVID-19 patients who displayed GI symptoms and those who did not. Compared with other acute phase markers, both fecal and serum calprotectin were superior in identifying COVID-19 patients who progressed to severe illness. Although the progression of COVID-19 disease is marked by an elevation of fecal and serum calprotectin, gastrointestinal symptoms or diarrhea were not correlated with calprotectin increase level.

Positive association between severity of COVID-19 infection and liver damage: a systematic review and meta-analysis.

AIM: The current study aimed to report a pooled analysis of the association of the circulating levels of liver enzymes and total bilirubin with severe and non-severe COVID-19. BACKGROUND: The ongoing coronavirus outbreak is an important threat to health worldwide. Epidemiological data representing greater risk of liver failure in patients infected with Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). METHODS: Electronic databases were comprehensively searched using Medline, ISI Web of Science, EMBASE, and the Cochrane Library up to July 2020. Outcomes from each relevant study were pooled using a random-effects model. Heterogeneity was analyzed by Q test and I2 statistics. Sensitivity analysis was also evaluated. RESULTS: A total of 24 studies were included (4,246 patients) in this study. We found a significant association of COVID-19 severity with increased levels of ALT [SMD: 1.40 U/L; 95% CI (0.93, 1.88); P < 0.05, I2 = 96.5%, P Heterogenity = 0.000 ], AST [SMD: 2.11 U/L; 95% CI (1.40, 2.83); P < 0.05, I2 = 97.9%, P Heterogenity = 0.000], LDH [SMD: 3.88 U/L; 95% CI (2.70, 5); P < 0.05, I2 = 98.7%, P Heterogenity = 0.000] and TBil [SMD: 1.08 mol/L; 95% CI (0.44, 1.72); P = 0.001, I2 = 97.7, P Heterogenity = 0.000], whereas, ALP values [SMD: 0.31; 95% CI (-1.57, 2.20); P = 0.74] was not significant between severe and non-severe COVID-19 patients. Moreover, elevated liver enzymes were found more in males [OR: 1.52, (95% CI 1.26, 1.83), P < 0.05] with severe COVID-19 infection than in females. CONCLUSION: The alterations of liver function indexes caused by SARS-CoV-2 infection suggested a potential prognosis biomarker for screening of severe patients at early stages of the disease.

Nicotinamide Phosphoribosyltransferase Knockdown Leads to Lipid Accumulation in HepG2 Cells through The SIRT1-AMPK Pathway.

OBJECTIVE: Nicotinamide phosphoribosyltransferase (NAMPT), which is responsible for biosynthesis of nicotinamide adenine dinucleotide (NAD), has a regulatory role in cellular metabolism and thus, might be implicated in non-alcoholic fatty liver disease (NAFLD). This study aimed to show how NAMPT down-regulation in liver cells influences lipid metabolism and sirtiun 1 (SIRT1), as the main NAD-dependent deacetylase enzyme. MATERIALS AND METHODS: In this experimental study, HepG2 cells were transfected with NAMPT siRNA and hepatic triglyceride (TG) content and SIRT1 deacetylase activity were measured by colorimetric and fluorometric methods, respectively. Gene expression of fatty acid synthase (FAS) and sterol regulatory element-binding protein-1c (SREBP- 1c) was evaluated by real-time polymerase chain reaction (PCR). Total protein level and the phosphorylated form of acetyl-CoA carboxylase (ACC) and AMP-activated protein kinase (AMPK) were also investigated by western blotting. RESULTS: Knockdown of NAMPT significantly promoted the accumulation of TG in HepG2 cells, accompanied by a remarkable decline in SIRT1 deacetylase activity. A significant rise in the gene expression of two key lipogenic factors, FAS and SREBP-1c was also observed. These effects were also accompanied by decreased phosphorylation of ACC and AMPK. On the other hand, treatment of transfected cells with either NAD, as the SIRT1 substrate or resveratrol, as the SIRT1 activator reversed the outcomes. CONCLUSION: These results demonstrated a protective role for NAMPT against NAFLD and its involvement in the regulation of de novo lipogenesis through the SIRT1/AMPK pathway.

Hesperetin is a potent bioactivator that activates SIRT1-AMPK signaling pathway in HepG2 cells.

Sirtuin 1 (SIRT1) is a deacetylase enzyme that plays crucial roles in controlling many cellular processes and its downregulation has been implicated in different metabolic disorders. Recently, several polyphenols have been considered as the effective therapeutic approaches that appear to influence SIRT1. The main goal of this study was to evaluate the effect of hesperetin, a citrus polyphenolic flavonoid, on SIRT1 and AMP-activated kinase (AMPK). HepG2 cells were treated with hesperetin in the presence or absence of EX-527, a SIRT1 specific inhibitor, for 24 h. Resveratrol was used as a positive control. SIRT1 gene expression, protein level, and activity were measured by RT-PCR, Western blotting, and fluorometric assay, respectively. AMPK phosphorylation was also determined by Western blotting. Our results indicated a significant increase in SIRT1 protein level and activity as well as an induction of AMPK phosphorylation by hesperetin. These effects of hesperetin were abolished by EX-527. Furthermore, hesperetin reversed the EX-527 inhibitory effects on SIRT1 protein expression and AMPK phosphorylation. These findings suggest that hesperetin can be a novel SIRT1 activator, even stronger than resveratrol. Therefore, the current study may introduce hesperetin as a new strategy aimed at upregulation SIRT1-AMPK pathway resulting in various cellular processes regulation.

Investigating the effect of visfatin on ERalpha phosphorylation (Ser118 and Ser167) and ERE-dependent transcriptional activity.

Obesity is associated with higher postmenopausal breast cancer incidence. Visfatin level alteration is one of the mechanisms by which obesity promotes cancer. Ligand-independent activation of estrogen receptor alpha (ERα) is also associated with carcinogenesis. The activity of ERα is modulated through phosphorylation on multiple sites by a number of protein kinases. Here we investigated the effect of visfatin as a novel adipocytokine on the phosphorylation and activity of ERα in MCF-7 breast cancer cells. We showed that exogenous administration of visfatin significantly increased the phosphorylation of ERα at serine 118 (Ser118) and 167 (Ser167) residues. Visfatin-induced Ser118 phosphorylation was diminished after treatment of cells with U0126 (MEK1/2 inhibitor). Furthermore, our results showed that visfatin-induced Ser167 phosphorylation is mediated through both MAPK and PI3K/Akt signaling pathways. Inhibition of the enzymatic activity of visfatin by FK866 had no effect on phosphorylation of ERα. We also showed that visfatin enhanced the estrogen response element (ERE)-dependent activity of ER in the presence of 17-ß estradiol (E2). Additional study on T47D cells showed that visfatin also increased Ser118 and Ser167 phosphorylation of ERα and enhanced ERE-dependent activity in the presence of E2 in these cells.