Background: Treatment with the chemotherapy drug doxorubicin (DOX) may lead to toxicities that affect non-cancer cells including the liver. Supplementing the diet with creatine (Cr) has been suggested as a potential intervention to minimize DOX-induced side effects, but its effect in alleviating DOX-induced hepatoxicity is currently unknown. Therefore, we aimed to examine the effects of Cr supplementation on DOX-induced liver damage. Methods: Male Sprague-Dawley rats were fed a diet supplemented with 2% Cr for four weeks, 4% Cr for one week followed by 2% Cr for three more weeks, or control diet for four weeks. Animals then received either a bolus i.p. injection of DOX (15 mg/kg) or saline as a placebo. Animals were then sacrificed five days-post injection and markers of hepatoxicity were analyzed using the liver-to-body weight ratio, aspartate transaminase (AST)-to- alanine aminotransferase (ALT) ratio, alkaline phosphatase (ALP), lipemia, and T-Bilirubin. In addition, hematoxylin and eosin (H&E) staining, Picro-Sirius Red staining, and immunofluorescence staining for CD45, 8-OHdG, and β-galactosidase were performed to evaluate liver morphology, fibrosis, inflammation, oxidative stress, and cellular senescence, respectively. The mRNA levels for biomarkers of liver fibrosis, inflammation, oxidative stress, and senescence-related genes were measured in liver tissues. Chromosomal stability was evaluated using global DNA methylation ELISA. Results: The ALT/AST ratio and liver to body weight ratio tended to increase in the DOX group, and Cr supplementation tended to attenuate this increase. Furthermore, elevated levels of liver fibrosis, inflammation, oxidative stress, and senescence were observed with DOX treatment, and Cr supplementation prior to DOX treatment ameliorated this hepatoxicity. Moreover, DOX treatment resulted in chromosomal instability (i.e., altered DNA methylation profile), and Cr supplementation showed a tendency to restore chromosomal stability with DOX treatment. Conclusion: The data suggest that Cr protected against DOX-induced hepatotoxicity by attenuating fibrosis, inflammation, oxidative stress, and senescence.

Cholangiocarcinoma (CCA) is a rare and highly lethal disease with few effective treatment options. Cannabinoids, cannabidiol (CBD) and cannabigerol (CBG) are non-psychedelic components extracted from cannabis. These non-psychoactive compounds have shown anti-proliferative potential in other tumor models; however, the efficacy of CBD and CBG in CCA is unknown. Furthermore, two cell death pathways are implicated with CBD resulting in autophagic degeneration and CBG in apoptosis. HuCC-T1 cells, Mz-ChA-1 cells (CCA cell lines) and H69 cells (immortalized cholangiocytes), were treated with CBD and CBG for 24 to 48 h. The influence of these cannabinoids on proliferation was assessed via MTT assay. Apoptosis and cell cycle were evaluated via Annexin-V apoptosis assay and propidium iodide, respectively. The expression of proliferation biomarker Ki-67, apoptosis biomarker BAX, and autophagic flux biomarkers LC3b and LAMP1 were evaluated via immunofluorescence. Cell migration and invasion were evaluated via wound healing assay and trans-well migration invasion assays, respectively. The colony formation was evaluated via colony formation assay. In addition, the expression of autophagy gene LC3b and apoptosis genes BAX, Bcl-2, and cleaved caspase-3 were evaluated via Western blot. CBD and CBG are non-selective anti-proliferative agents yielding similar growth curves in CCA; both cannabinoids are effective, yet CBG is more active at lower doses. Low doses of CBD and CBG enhanced immortalized cholangiocyte activity. The reduction in proliferation begins immediately and occurs maximally within 24 h of treatment. Moreover, a significant increase in the late-stage apoptosis and a reduction in the number of cells in S stage of the cell cycle indicates both CBD and CBG treatment could promote apoptosis and inhibit mitosis in CCA cells. The fluorescent expression of BAX and LC3b was significantly enhanced with CBD treatment when compared to control. LAMP1 and LC3b colocalization could also be observed with CBD and CBG treatment indicating changes in autophagic flux. A significant inhibition of migration, invasion and colony formation ability was shown in both CBD and CBG treatment in CCA. Western blot showed an overall decrease in the ratio of anti-apoptotic protein Bcl-2 with respect to pro-apoptotic protein BAX with CBG treatment. Furthermore, CBD treatment enhanced the expression of Type II cell death (autophagic degeneration) protein LC3b, which was reduced in CBG-treated CCA cells. Meanwhile, CBG treatment upregulated Type I cell death (programmed apoptosis) protein cleaved caspase-3. CBD and CBG are effective anti-cancer agents against CCA, capable of inhibiting the classic hallmarks of cancer, with a divergent mechanism of action (Type II or Type I respectively) in inducing these effects.

Background and aim Doxorubicin is a chemotherapeutic agent that is widely used for several malignancies. It is known to have severe side effects such as hepatotoxicity, cardiotoxicity and increase in nitric oxide concentrations in skeletal muscles. On the other hand, creatine monohydrate is a supplementation used as a therapeutic agent and plays a significant role as regenerative agent. So far, there is not enough information about the effect of doxorubicin on the overall liver functionality and how creatine monohydrate helps in easing the hepatotoxicity; therefore, our aim is to investigate the role of creatine monohydrate supplementation in alleviating the hepatotoxicity caused by doxorubicin. Methods Sprague‐Dawley rats were fed rodent chow, 2% creatine, 4% followed by 2% creatine supplementation for our weeks; and 15 mg/kg doxorubicin was given once the day before they were sacrificed. Peripheral blood and liver were harvested and snap frozen. Serum chemistry was obtained to evaluate the liver function by looking at the levels of Lipemia, T‐Bilirubin, AST, ALP and ALT. Liver damage was evaluated by using Hematoxylin and Eosin staining; whereas liver fibrosis was evaluated by Sirius Red staining. RT‐PCR was performed to assess the gene expression of fibrotic genes, proliferative genes, oxidative stress and apoptosis‐related genes at the transcription level, and western blotting was performed to measure the gene expression at the protein level. Results There was no significant difference found in the serum chemistry in the treatment groups when compared to the control group. However, the histology showed an increase in inflammation and fibrosis in the doxorubicin group when compared to the control, where there was a decrease observed in the group treated with 4% creatine followed by 2% creatine. A significant increase in the gene expression of Bax, CK‐19 and Col1‐a1 has been observed in the 2% creatine + doxorubicin. Further, there was a decrease in the protein expression of CASP3 in the 2% creatine + doxorubicin and 4%/2% creatine + doxorubicin when compared to the doxorubicin group. Conclusion Based on the collected data and observed behavior of groups, we conclude that the gradual dose of creatine monohydrate supplementation does alleviate the hepatotoxicity caused by doxorubicin. Support or Funding Information Graduate Student Association at the University of Northern Colorado

Non-Alcoholic Steatohepatitis (NASH) is the progressive form of Non-Alcoholic Fatty Liver Disease (NAFLD). NASH is distinguished by severe hepatic fibrosis and inflammation. The plant-derived, non-psychotropic compound cannabigerol (CBG) has potential anti-inflammatory effects similar to other cannabinoids. However, the impact of CBG on NASH pathology is still unknown. This study demonstrated the therapeutic potential of CBG in reducing hepatic steatosis, fibrosis, and inflammation. Methods: 8-week-old C57BL/6 male mice were fed with methionine/choline deficient (MCD) diet or control (CTR) diets for five weeks. At the beginning of week 4, mice were divided into three sub-groups and injected with either a vehicle, a low or high dose of CBG for two weeks. Overall health of the mice, Hepatic steatosis, fibrosis, and inflammation were evaluated. Results: Increased liver-to-body weight ratio was observed in mice fed with MCD diet, while a low dose of CBG treatment rescued the liver-to-body weight ratio. Hepatic ballooning and leukocyte infiltration were decreased in MCD mice with a low dose of CBG treatment, whereas the CBG treatment did not change the hepatic steatosis. The high dose CBG administration increased inflammation and fibrosis. Similarly, the expression of cannabinoid receptor (CB)1 and CB2 showed decreased expression with the low CBG dose but not with the high CBG dose intervention in the MCD group and were co-localized with mast cells. Additionally, the decreased mast cells were accompanied by decreased expression of transforming growth factor (TGF)-β1. Conclusions: Collectively, the low dose of CBG alleviated hepatic fibrosis and inflammation in MCD-induced NASH, however, the high dose of CBG treatment showed enhanced liver damage when compared to MCD only group. These results will provide pre-clinical data to guide future intervention studies in humans addressing the potential uses of CBG for inflammatory liver pathologies, as well as open the door for further investigation into systemic inflammatory pathologies.