Nonalcoholic fatty liver disease (NAFLD) is one of the etiologies that contribute to hepatocellular carcinoma (HCC), and chronic inflammation is one of the proposed mediators of HCC. Because necroptosis is a cell death pathway that induces inflammation, we tested whether necroptosis-induced inflammation contributes to the progression of NAFLD to HCC in a mouse model of diet-induced HCC. Male and female wild-type (WT) mice and mouse models where necroptosis is blocked (Ripk3−/− or Mlkl−/− mice) were fed either a control diet, choline-deficient low-fat diet or choline-deficient high-fat diet. Blocking necroptosis reduced markers of inflammation [proinflammatory cytokines (TNFα, IL6, and IL1β), F4/80+ve macrophages, CCR2+ve infiltrating monocytes], inflammation-associated oncogenic pathways (JNK, PD-L1/PD-1, β-catenin), and HCC in male mice. We demonstrate that hepatic necroptosis promotes recruitment and activation of liver macrophages leading to chronic inflammation, which in turn trigger oncogenic pathways leading to the progression of NAFLD to HCC in male mice. Whereas in female mice, blocking necroptosis reduced HCC independent of inflammation. Our data show a sex-specific difference in the development of inflammation, fibrosis, and HCC in WT mice. However, blocking necroptosis reduced HCC in both males and females without altering liver fibrosis. Thus, our study suggests that necroptosis is a valid therapeutic target for NAFLD-mediated HCC. Implications: Necroptosis is a major contributor to hepatic inflammation that drives the progression of NAFLD to HCC and therefore represents a valid target for NAFLD-mediated HCC.

Chronic inflammation is a key player in metabolic dysfunction-associated fatty liver disease (MAFLD) progression. Necroptosis, an inflammatory cell death pathway, is elevated in MAFLD patients and mouse models, yet its role is unclear due to the diverse mouse models and inhibition strategies. In our study, we inhibited necroptosis by targeting mixed lineage kinase domain-like pseudokinase (MLKL), the terminal effector of necroptosis, in a high-fat, high-fructose, high-cholesterol (HFHFrHC) mouse model of diet-induced MAFLD. Despite the HFHFrHC diet upregulating MLKL (2.5-fold), WT mice livers showed no increase in necroptosis markers or associated proinflammatory cytokines. Surprisingly, Mlkl^−/− mice experienced exacerbated liver inflammation without protection from diet-induced liver damage, steatosis, or fibrosis. In contrast, Mlkl^+/− mice showed a significant reduction in these parameters that was associated with elevated Pparα and Pparγ levels. Both Mlkl^−/− and Mlkl^+/− mice on the HFHFrHC diet resisted diet-induced obesity, attributed to the increased beiging, enhanced oxygen consumption, and energy expenditure due to adipose tissue, and exhibited improved insulin sensitivity. These findings highlight the tissue-specific effects of MLKL on the liver and adipose tissue, and they suggest a dose-dependent effect of MLKL on liver pathology.

This study was conducted to comparatively assess the anthelminthic activity of leaves, stem bark, and seeds of Carica papaya, in order to identify which of the plant parts possess the highest anthelminthic activity. Three concentrations of ethanolic and hydroethanolic extracts of the plant parts (1 mg/ml, 2.5 mg/ml, and 5 mg/ml) were prepared and tested against Pheretima posthuma using albendazole as the positive control and 0.9% normal saline solution as the negative control. Preliminary phytochemical investigation showed the presence of alkaloids, saponins, and reducing sugars of glycosides present in all the crude extracts of Carica papaya. Tannins were observed only in extracts of the leaves, while fixed oils were only present in the extracts of the seeds. The results of the anthelminthic activity testing indicated that all crude extracts prepared were more effective than albendazole in reducing paralysis time (p < 0.0001) and death time (p < 0.0001). It was further shown that the extracts from the seeds (SE and SHE) were more effective than the extracts from the stem bark and leaves both in reducing paralysis and death times. Fractionation of SE provided a fraction, SE_B, which was more effective than SE both in reducing paralysis and death times (p < 0.0001) and was established to contain fixed oils. The outcome of the current study has provided a scientific justification for the preference of the seeds of Carica papaya for the treatment of helminth infections and has shown that the fixed oils present in the seeds could be responsible for such activity.

An accurate and rapid reverse HPLC method has been developed and validated for the simultaneous quantification of lamivudine, nevirapine, and tenofovir disoproxil fumarate. Suitable separation was achieved on Phenomenex Synergi C18 (250 × 4.6 mm, 4 μm) using mobile phase, methanol (50%): ammonium acetate buffer (adjusted to pH 2.80) (40%): acetonitrile (10%) in an isocratic mode. The drugs were detected at 270 nm with a flow rate of 1.0 ml/min, and the retention times were found to be 3.26, 5.42, and 7.55 minutes for lamivudine, nevirapine, and tenofovir disoproxil fumarate, respectively. The developed method was validated per ICH guidelines. Good linearity was obtained within the concentration ranges of 10–59 µg/ml, 7–42 µg/ml, and 15–90 µg/ml with a correlation coefficient of not less than 0.990. The % RSD values for precision (intraday and interday) and accuracy studies were found to be less than 2%. The results obtained from quantitative analysis conform to USP content requirements for marketed tablet dosage forms, RICOVIR-LN, and tenofovir disoproxil fumarate/lamivudine tablets. The method is therefore useful for routine quality control of antiretroviral tablet dosage forms containing tenofovir disoproxil fumarate, lamivudine, and nevirapine.

Figure S2 shows body weight changes, liver weight changes, Transcript levels of Il1b and H&E staining for showing KC population

List of Differentially Expressed Genes from RNA Seq analysis