In- vitro Hepatoprotective Action of the Crude Extracts of Luffa amara (Whole Fruit) and Rheum emodi (Rhizomes) in CCl4 Intoxicated Rat Hepatocytes

Aim: The objective of this in-vitro study involves evaluating the protective action of the extracts of L. amara (LA) (whole fruits including seeds) and R. emodi (RE) (rhizomes) at various concentrations on isolated primary rat hepatocytes. Methods: The pulverised dried whole fruits of L. amara (LA) and rhizomes of R.emodi (RE) were extracted successively with petroleum ether (PE), ethanol (EE) and distilled water (AE) and vacuum dried. These extracts of LA petroleum ether (PE), ethanolic (EE) and aqueous (AE) extracts and RE obtained were subjected to in vitro studies at doses of 25, 75, 100, and 150 µg/ml and silymarin (250 µg/ml) in CCl4 (1%) intoxicated primary hepatocytes monolayer cultures the hepatoprotective action of all the extracts of both plants at different doses was carried out using isolated rat hepatocytes which were subjected to CCl4 intoxication followed by estimating/ measuring the changes in serum biochemical markers – SGPT, SGOT, ALP, Total proteins (TP), total bilirubin (TB), albumin (ALB) and triglycerides (TGL). Results: Hepatoprotective activity against CCl4 was demonstrated in the rat primary monolayer hepatocyte culture using MMT assay with the ethanolic extracts of both plants showing more hepatocyte protective action compared to the aqueous and petroleum ether extracts by reducing the elevated serum marker levels. Alcoholic and aqueous extracts were found to express more protective action towards CCl4 intoxicated isolated primary rat hepatocytes in a dose dependant manner. Conclusion: Based on the result, it is suggested that the extract with the most hepatocyte protective action at a dose of 150µg is LA ethanolic extract (viability=88.24%), followed by LA aqueous extract (viability=84.31%), RE ethanolic extract (viability=88.24%) and RE aqueous extract (viability=88.24%) - which are comparable to the reference silymarin with viability at 92.15%. the petroleum ether extracts of both plants showed least hepatic cell viability with LA pet ether extract at 49.02% and RE pet ether extract at 47.85%

In Vitro Proliferation and Long-Term Preservation of Functional Primary Rat Hepatocytes in Cell Fibers

Primary hepatocytes are essential cellular resource for drug screening and medical transplantation. Since culture systems for them have already succeeded in reconstituting the biomimetic microenvironment, acquiring additional capabilities both to expand primary hepatocytes and to handle them easily would be expected as progress to the next stage. This paper describes a culture system for primary rat hepatocytes that is equipped with scalability and handleability relying on cell fiber technology. Cell fibers are cell-laden core-shell hydrogel microfibers; in the core regions, cells are embedded in extracellular matrix proteins, cultured three-dimensionally, and exposed to soluble growth factors in the culture medium through the hydrogel shells. By encapsulating primary rat hepatocytes within cell fibers, we first demonstrated they increase in number while keeping their viability and their hepatic specific functions for up to thirty days of subsequent culture. Then, we demonstrated the potency of the primary rat hepatocytes that proliferate in cell fibers not only as cell-based sensors to detect drugs that damage hepatic functions and hepatocellular processes but also as transplants to improve the plasma albumin concentrations of congenital analbuminemia. Therefore, our culture system could serve for innovating strategies and promising developments in applying primary hepatocytes to both pharmaceutical and medical fields.

In Vitro Proliferation and Long-Term Preservation of Functional Primary Rat Hepatocytes in Cell Fibers

Primary hepatocytes are essential cellular resource for drug screening and medical transplantation. Since culture systems for them have already succeeded in reconstituting the biomimetic microenvironment, acquiring additional capabilities both to expand primary hepatocytes and to handle them easily would be expected as progress to the next stage. This paper describes a culture system for primary rat hepatocytes that is equipped with scalability and handleability relying on cell fiber technology. Cell fibers are cell-laden core-shell hydrogel microfibers; in the core regions, cells are embedded in extracellular matrix proteins, cultured three-dimensionally, and exposed to soluble growth factors in the culture medium through the hydrogel shells. By encapsulating primary rat hepatocytes within cell fibers, we first demonstrated they increase in number while keeping their viability and their hepatic specific functions for up to thirty days of subsequent culture. Then, we demonstrated the potency of the primary rat hepatocytes that proliferate in cell fibers not only as cell-based sensors to detect drugs that damage hepatic functions and hepatocellular processes but also as transplants to improve the plasma albumin concentrations of congenital analbuminemia. Therefore, our culture system could serve for innovating strategies and promising developments in applying primary hepatocytes to both pharmaceutical and medical fields.

CHOP Regulates Endoplasmic Reticulum Stress-Mediated Hepatoxicity Induced by Monocrotaline

Monocrotaline (MCT), a pyrrolizidine alkaloid, is the major toxin in Crotalaria, which causes cell apoptosis in humans and animals. It has been reported that the liver is a vulnerable target of MCT. However, the exact molecular mechanism of the interaction between endoplasmic reticulum (ER) stress and liver injury induced by MCT is still unclear. In this study, the cytotoxicity of MCT on primary rat hepatocytes was analyzed by a CCK-8 assay and Annexin V-FITC/PI assay. Protein expression was detected by western blotting and immunofluorescence staining. As a result, MCT significantly decreased the cell viability and mediated the apoptosis of primary rat hepatocytes. Meanwhile, MCT could also induce ER stress in hepatocytes, indicated by the expression of ER stress-related proteins, including GRP78, p-IRE1α, ATF6, p-eIF2α, ATF4, and CHOP. Pretreatment with 4-PBA, an inhibitor of ER stress, or knockdown of CHOP by siRNA could partly enhance cell viability and relieve the apoptosis. Our findings indicate that ER stress is involved in the hepatotoxicity induced by MCT, and CHOP plays an important role in this process.

The Influence of Coumestrol on Sphingolipid Signaling Pathway and Insulin Resistance Development in Primary Rat Hepatocytes

Coumestrol is a phytoestrogen widely known for its anti-diabetic, anti-oxidant, and anti-inflammatory properties. Thus, it gets a lot of attention as a potential agent in the nutritional therapy of diseases such as obesity and type 2 diabetes. In our study, we evaluated whether coumestrol affects insulin resistance development via the sphingolipid signaling pathway in primary rat hepatocytes. The cells were isolated from the male Wistar rat’s liver with the use of collagenase perfusion. Next, we incubated the cells with the presence or absence of palmitic acid and/or coumestrol. Additionally, some groups were incubated with insulin. The sphingolipid concentrations were assessed by HPLC whereas the expression of all the proteins was evaluated by Western blot. Coumestrol markedly reduced the accumulation of sphingolipids, namely, ceramide and sphinganine through noticeable inhibition of the ceramide de novo synthesis pathway in insulin-resistant hepatocytes. Moreover, coumestrol augmented the expression of fatty acid transport proteins, especially FATP5 and FAT/CD36, which also were responsible for excessive sphingolipid accumulation. Furthermore, coumestrol altered the sphingolipid salvage pathway, which was observed as the excessive deposition of the sphingosine-1-phosphate and sphingosine. Our study clearly showed that coumestrol ameliorated hepatic insulin resistance in primary rat hepatocytes. Thus, we believe that our study may contribute to the discovery of novel preventive and therapeutic methods for metabolic disorders.