Synergistic Effects of Vitis vinifera L. and Centella asiatica against CCl4-Induced Liver Injury in Mice.

Liver injury can be acute or chronic, resulting from a variety of factors, including viral hepatitis, drug overdose, idiosyncratic drug reaction, or toxins, while the progression of pathogenesis in the liver rises due to the involvement of numerous cytokines and growth factor mediators. Thus, the identification of more effective biomarker-based active phytochemicals isolated from medicinal plants is a promising strategy to protect against CCl4-induced liver injury. Vitis vinifera L. (VE) and Centella asiatica (CE) are well-known medicinal plants that possess anti-inflammatory and antioxidant properties. However, synergism between the two has not previously been studied. Here, we investigated the synergistic effects of a V. vinifera L. (VE) leaf, C. asiatica (CE) extract combination (VCEC) against CCl4-induced liver injury. Acute liver injury was induced by a single intraperitoneal administration of CCl4 (1 mL/kg). VCEC was administered orally for three consecutive days at various concentrations (100 and 200 mg/kg) prior to CCl4 injection. The extent of liver injury and the protective effects of VCEC were evaluated by biochemical analysis and histopathological studies. Oxidative stress was evaluated by measuring malondialdehyde (MDA) and glutathione (GSH) levels and Western blotting. VCEC treatment significantly reduced serum transaminase levels (AST and ALT), tumor necrosis factor-α (TNF-α), and reactive oxygen species (ROS). CCl4- induced apoptosis was inhibited by VCEC treatment by reducing cleaved caspase-3 and Bcl2-associated X protein (Bax). VCEC-treated mice significantly restored cytochrome P450 2E1, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) expression in CCl4-treated mice. In addition, VCEC downregulated overexpression of proinflammatory cytokines and hepatic nuclear factor kappa B (NF-κB) and inhibited CCl4-mediated apoptosis. Collectively, VCEC exhibited synergistic protective effects against liver injury through its antioxidant, anti-inflammatory, and antiapoptotic ability against oxidative stress, inflammation, and apoptosis. Therefore, VCEC appears promising as a potential therapeutic agent for CCl4-induced acute liver injury in mice.

Hepatoprotective Effects of a Natural Flavanol 3,3'-Diindolylmethane against CCl4-Induced Chronic Liver Injury in Mice and TGFß1-Induced EMT in Mouse Hepatocytes via Activation of Nrf2 Cascade.

Hepatic fibrosis is a form of irregular wound-healing response with acute and chronic injury triggered by the deposition of excessive extracellular matrix. Epithelial-mesenchymal transition (EMT) is a dynamic process that plays a crucial role in the fibrogenic response and pathogenesis of liver fibrosis. In the present study, we postulated a protective role of 3,3'-diindolylmethane (DIM) against TGF-ß1 mediated epithelial-mesenchymal transition (EMT) in vitro and carbon tetrachloride (CCl4)-induced liver fibrosis in mice. TGF-ß1-induced AML-12 hepatocyte injury was evaluated by monitoring cell morphology, measuring reactive oxygen species (ROS) and mitochondrial membrane potential, and quantifying apoptosis, inflammatory, and EMT-related proteins. Furthermore, CCl4-induced liver fibrosis in mice was evaluated by performing liver function tests, including serum ALT and AST, total bilirubin, and albumin to assess liver injury and by performing H&E and Sirius red staining to determine the degree of liver fibrosis. Immunoblotting was performed to determine the expression levels of inflammation, apoptosis, and Nrf2/HO-1 signaling-related proteins. DIM treatment significantly restored TGF-ß1-induced morphological changes, inhibited the expression of mesenchymal markers by activating E-cadherin, decreased mitochondrial membrane potential, reduced ROS intensity, and upregulated levels of Nrf2-responsive antioxidant genes. In the mouse model of CCl4-induced liver fibrosis, DIM remarkably attenuated liver injury and liver fibrosis, as reflected by the reduced ALT and AST parameters with increased serum Alb activity and fewer lesions in H&E staining. It also mitigated the fibrosis area in Sirius red and Masson staining. Taken together, our results suggest a possible molecular mechanism of DIM by suppressing TGF-ß1-induced EMT in mouse hepatocytes and CCl4-induced liver fibrosis in mice.

Hispidin analogue davallialactone attenuates carbon tetrachloride-induced hepatotoxicity in mice.

In this study the protective effects of davallialactone (1), isolated from Inonotus xeranticus, have been examined against carbon tetrachloride (CCl4-induced acute liver injury. Mice received subcutaneous injection of 1 (2.5, 5, and 10 mg/kg) for three days before CCl4 injection (1 mg/kg). Protection from liver injury by 1 was confirmed by the observation of decreased serum transaminases and diminished necrosis of liver tissue. Reduced hepatic injury was very similar to that observed with silymarin, a known hepatoprotective drug used in this work for comparison. The groups treated with 1 had reduced reactive oxygen species (ROS), reduced serum malonyldialdehyde levels, and increased levels of liver Cu/Zn superoxide dismutase, as compared to the CCl4 control group. The expression of heme oxygenase-1 in the liver tissue was increased and the activity of liver cytochrome P4502E1 was restored in the mice treated with 1. In addition, levels of serum tumor necrosis factor-alpha (TNF-α), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2), numbers of macrophage, and cleaved caspase-3-positive hepatocytes were reduced in the groups treated with 1. These findings suggest that davallialactone has protective effects against CCl4-induced acute liver injury, and this protection is likely due to the suppression of ROS-induced lipid peroxidation and inflammatory response.

Overexpression of peptidyl-prolyl isomerase Pin1 attenuates hepatocytes apoptosis and secondary necrosis following carbon tetrachloride-induced acute liver injury in mice.

Pin1, a member of the parvulin family of PPIase enzymes, plays a crucial role in the post phosphorylation regulation that governs important roles in the cell signaling mechanism and regulates a variety of cellular events. In this study, we investigated the role of Pin1 in carbon tetrachloride (CCl(4))-induced apoptosis and necrosis of hepatocytes during acute liver injury of mice. An in vivo study was done with the overexpression of Pin1 in the mouse liver; using Pin1-adenoviruse (ad-Pin1) followed by CCl(4) injection to induce acute liver injury. Pin1 overexpression in the liver of the experimental mice attenuated acute liver injury induced by CCl(4) . Serum aminotransferases and the number of apoptotic cells were decreased compared to those of control virus injected mice. In addition, Pin1 overexpression increased NF-kB activity, as evidenced by increased DNA binding. In conclusion, Pin1 reduces acute liver injury of mice due to CCl(4) by modulating apoptotic signals and by increasing NF-kB activity.

C-reactive Protein to Albumin Ratio among Patients Admitted to Intensive Care Unit of a Tertiary Care Hospital: A Descriptive Crosssectional Study.

INTRODUCTION: Sepsis is the most common cause of mortality among patients admitted to intensive care unit. There is emerging evidence on the role of C-reactive protein to albumin ratio (C-reactive protein/Albumin) in predicting outcomes in patients with critical illness and sepsis, admitted to intensive care unit. We aimed to find out the median value of C-reactive protein/Albumin ratio among patients admitted to intensive care unit of a tertiary care hospital. METHODS: We conducted a descriptive cross-sectional study of 110 critically ill patients (>18 years old) admitted to intensive care unit of Dhulikhel Hospital from April, 2014 to June, 2016. The ethical approval (Reference number.51/16) was obtained from Institutional Review Committee at Kathmandu University School of Medical Sciences. C-reactive protein/albumin ratio was calculated from records of patients admitted to the intensive care unit. Convenience sampling was done. Data were entered into Microsoft Excel and analysed using Statistical Package for Social Sciences version 20. Point estimate at 95% Confidence Interval was calculated along with frequencies and percentages for binary data. RESULTS: Among 110 patients admitted to the intensive care unit, the median value of C-reactive protein/Albumin ratio was found to be 3.4 (Interquartile range: 3.1-4.5). Out of these patients, 44 (39.5%) patients were septic and their median C-reactive protein/albumin ratio was 3.4 (Interquartile range: 3.1-4.5). CONCLUSIONS: Our study showed higher median C-reactive protein /Albumin similar to other studies. Sepsis is a common finding among patients admitted to intensive care unit. Monitoring of C-reactive protein/albumin level in a patient admitted to intensive care unit could be useful for stratifying patients with a high risk of developing sepsis.

Factors Affecting Turnaround Time in the Clinical Laboratory of the Kathmandu University Hospital, Nepal.

BACKGROUND: The turnaround time (TAT) as defined by most of the laboratories is the time interval between the specimens received in the laboratory to the time of reports dispatched with verification. Nearly 80% of hospital-attached clinical laboratories receive complaints about delayed TAT. Reporting in time is a crucial indicator of quality services along with accurate, precise and reliable reports, thus each clinical laboratory should identify affecting factors to eliminate them for the enhancement of quality services. METHODOLOGY: Dhulikhel Hospital-Kathmandu University Hospital is a tertiary care hospital, where this observational descriptive study was conducted in 2017. Requested tests received on database in the Department of Clinical Biochemistry Laboratory along with test requisition form (TRF) were carefully screened for any possible error. When analysis of individual patient's tests was completed, results of individual parameters were entered in the database manually. TAT was calculated as a time period between specimens received to analysis completed. Once test analysis has completed it was immediately followed by verification. RESULTS: A total of 36,108 patients' reports generated from the Department of Clinical Biochemistry Laboratory during study period were analyzed. Nearly 36% of reports exceeded the predefined TAT in case of stat tests, while around 7% of reports were out of predefined TAT in case of routine tests. Among prolonged TAT, around 75% of reports were delayed due to various extra analytical reasons and approximately 48% of total delayed reports were found only due to error by cash unit. CONCLUSION: The major reasons of delayed laboratory reports were due to time consumed to fix the pre-analytical errors created by other departments rather than laboratory itself. Cash unit alone has the highest degree of error in total testing process and it is the most significant factor for prolonged TAT. However reasons for prolonged TAT may vary with hospital to hospital depending upon different factors.

Prevalence and Factors Associated with Microalbuminuria among Type 2 Diabetic Patients : A Hospital Based Study.

INTRODUCTION: Microalbuminuria is the earliest clinical evidence of diabetic nephropathy. However, prevalence and associated factors with microalbuminuria among type 2 diabetic patients has been understudied area of research in Nepalese context. This study aimed to determine the prevalence and factors associated with microalbuminuria among type 2 diabetic patients. METHODS: This study was a hospital-based cross-sectional study. Blood samples for serum creatinine, Hemoglobin A1C, Fasting blood sugar and urine sample for microalbumin and urine creatinine were collected and analyzed using validated and standardized tools from a total of 400 Type 2 diabetic patients in Devdaha Medical College and Teaching Hospital, Rupandehi, Nepal from August 2014 to September 2017. Microalbuminuria was defined as urinary albumin-to-creatinine ratio greater than 30 and less than300 µg /mg of creatinine Results: Of 400 type 2 diabetic patients, 186 (46.5%) had microalbuminuria. The mean values of FBS, HbA1C, serum creatinine, microalbumin, microalbumin/urine creatinine ratio were higher in microalbuminuria group. Microalbuminuria was significantly positively correlated with duration of diabetes, FBS, HbA1C, serum creatinine, microalbumin, microalbumin/ urine creatinine, systolic blood pressure and diastolic blood pressure (P< 0.01). CONCLUSIONS: Our study demonstrated that nearly half of the type 2 diabetic patients had microalbuminuria. Our results emphasize to increase to accessibility to microalbuminuria testing for all the type 2 diabetic patients and bring them under medical supervision to reduce the unwanted complications of diabetes mellitus.

Involvement of prolyl isomerase PIN1 in the cell cycle progression and proliferation of hepatic oval cells.

Liver regenerates remarkably after toxic injury or surgical resection. In the case of failure of resident hepatocytes to restore loss, repopulation is carried out by induction, proliferation, and differentiation of the progenitor cell. Although, some signaling pathways have been verified to contribute oval cell-mediated liver regeneration, role of Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1(Pin1) in the oval cells proliferation is unknown. In the present study, we evaluate the role of Pin1 in oval cells proliferation. In our study, the expression of Pin1 in the mice liver increased after three weeks feeding of 3, 5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) diet along with the proliferation of oval cells. The expression of Pin1 was higher in oval cells compared to the hepatocytes.Pin1 inhibition by Juglone reduced oval cell proliferation, which was restored to normal when oval cells were treated with IGF-1. Consistent with increased cell growth, expression of Pin1, ß-catenin and PCNA were increased in IGF-1 treated cells in a time dependent manner. In FACS analysis, siRNA-mediated knockdown of the Pin1 protein in the oval cells significantly increased the numbers of cells in G0/G1 phase. Furthermore, hepatocyte when treated with TGF-ß showed marked reduction in cell proliferation and expression of Pin1 whereas this effect was not seen in the oval cells treated with TGF-ß. In conclusion, Pin1 plays important role in the cell cycle progression and increase oval cells proliferation which may be crucial in chronic liver injury.

Pattern of thyroid dysfunction in patients with metabolic syndrome and its relationship with components of metabolic syndrome.

BACKGROUND: Thyroid dysfunction (TD) and metabolic syndrome (MetS) are known risk factors for atherosclerotic cardiovascular disease (ASCVD). TD is risk factor for ASCVD mediated by the effects of thyroid hormones on lipid metabolism and blood pressure hence the components of MetS. It is possible that coexistence of these two disease entities and unrecognized TD in patients with MetS might substantially increase ASCVD risk. Moreover, little is known about the relationship between TD and the components of MetS. Thus, the purpose of this study was to evaluate the pattern of TD in patients with MetS and its relationship with components of the MetS. METHODS: A total of 358 previously diagnosed patients with MetS were recruited in the study. The thyroid function test parameters were measured to classify TD at Dhulikhel Hospital-Kathmandu University Hospital, Dhulikhel, Nepal. Statistical analyses were performed using SPSS version 16.0 to evaluate pattern and relationship. RESULTS: The overall prevalence of TD in patients with MetS was 31.84% with high prevalence of subclinical hypothyroidism (29.32%). We found no evidence of a relationship between TD and components of MetS, although there was significant difference in waist circumference between four groups of TD. CONCLUSION: Patients with MetS had subclinical hypothyroidism greatly. Although there was no evidence of any relationship between thyroid status and all components of MetS, TD should be taken into account when evaluating and treating patients with MetS to reduce the impending risk.

Synthesis, characterization, and mineralization of polyamide-6/calcium lactate composite nanofibers for bone tissue engineering.

Polyamide-6 nanofibers containing calcium lactate (CL) on their surface were prepared by neutralization of lactic acid (LA) in core-shell structured polyamide-6/LA electrospun fibers. First, simple blending of LA with polyamide-6 solution was used for electrospinning which interestingly formed a thin LA layer around polyamide-6 nanofibers (core-shell structure) and then subsequent conversion of this LA into calcium lactate via neutralization using calcium base. FE-SEM and TEM images revealed that plasticizer capacity of LA led the formation of point-bonded structure due to the formation of shell layer of LA and core of polyamide-6. The bone formation ability of polyamide-6/calcium lactate composite fibers was evaluated by incubating in biomimetic simulated body fluid (SBF). The SBF incubation test confirmed the faster deposition of large amount of calcium phosphate around the composite polyamide-6/calcium lactate fibers compared to pristine polyamide-6. This study demonstrated a simple post electrospinning calcium compound coating technique of polymeric nanofibers for enhancing the bone biocompatibility of polyamide-6 fibers.

The establishment and characterization of immortal hepatocyte cell lines from a mouse liver injury model.

Hepatocytes are an important research tool used for numerous applications. However, a short life span and a limited capacity to replicate in vitro limit the usefulness of primary hepatocyte cultures. We have hypothesized that in vivo priming of hepatocyte could make them more susceptible to growth factors in the medium for continuous proliferation in vitro. Here, a novel approach used to establish hepatocyte cell lines that included hepatocyte priming in vivo prior to culture with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet was attempted. The cell line grew in a monolayer while maintaining a granular cytoplasm and a round nucleus. Electron microscopy displayed hepatocyte-like features including mitochondria, glycogen granules, and the presence of bile canaliculi. This cell line expressed many mature hepatocyte-specific genes including albumin, alpha1-antitrypsin, glucose 6-phosphatase, and tyrosine aminotransferase. Functional characteristic of hepatocytes like the ability to store glycogen, lipid, and synthesis of urea is well demonstrated by this cell line. These cells demonstrated anchorage dependent growth properties in soft agar and did not form tumors after transplantation into nude mice. This cell line can be sustained in culture for more than 100 passages (>1.5 years) without undergoing noticeable morphological changes or transformation. This novel method resulted in the establishment of an immortal, non-transformed hepatocyte cell line with functional characteristics that may aid research of cell metabolism, toxicology, and hepatocyte transplantation.

Spectrum of stones composition: a chemical analysis of renal stones of patients visiting NMCTH.

A general observation of clinicians suggests that the prevalence ofurolithiasis is fairly high in Kathmandu but so far no systematic study has been undertaken here to explore the etiopathogenesis of disease in this region. In this preliminary communication, we present herewith the qualitative composition of 47 renal stones collected from surgical patients admitted to NMCTH over a period of 13 months (July 2005 to July 2006). All stones were of mixed type. Calcium was present in all stones. Oxalate, phosphate and uric acid were present in 95.7%, 87.2% and 34.0% patients respectively. The probable composition, as construed from analysis, suggests that calcium oxalate stones are predominant. Strikingly, the prevalence was very high in e"20 yrs age group.