الفهرس 
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Abstract
Hepatocellular carcinoma (HCC) is a global public health issue in both developed and undeveloped nations. Disruption of hepatic mitochondrial homeostasis is considered a hallmark in HCC due to the hepatic mitochondria is a vital signaling and metabolic organ which maintain the balance of mitochondrial biogenesis, dynamics and mitophagy. Mitophagy is defined as an intracellular selective degradation pathway, required for the removal of dysfunctional mitochondria, misfolded proteins, and long-lived proteins in response to severe starvation or stress. It has been reported that there is a direct association between mitochondrial dysfunction and HCC. Recently, targeting mitophagy seems to affect anti-cancer drugs’ efficacy or chemo-sensitivity. Currently, used therapy is efficient against cancer, but it also induces cell death in healthy cells, resulting in significant side effects in patients.
Several natural chemicals with minimal toxicity and good selectivity for killing cancer cells have been employed as alternative cancer therapies, including HCC. Astaxanthin (ATX) is a dark red carotenoid and a type of short-chain antioxidant. It is considered a second-generation antioxidant that exhibits anti-carcinogenic properties including antioxidant, anti-inflammatory, and anti-mitochondrial dysfunction. In this regard, it is believed that its combination with conventional chemotherapies could be a safer approach to cancer prevention and therapeutic strategies. Astaxanthin either alone or in combination with conventional anti-cancer agents can efficiently sensitize tumor cells to a variety of drugs due to their effects on mitophagy activity.
This research was seeking to determine how ATX affected the expression of selected regulated genes that govern mitochondrial biogenesis, dynamics, and degradation paths. in a rat model of chemically induced HCC as an adjuvant chemotherapeutic drug with doxorubicin (DOX).
This study will be conducted on 30 male Wistar albino rats (weights ranging from 150-200g), 2-3 months old. Rats were given intraperitoneally (IP) a single dosage of DEN dissolved in normal saline for 200 milligrammes per kilogramme, and after two weeks, the HCC model was induced, subcutaneous injections of 3 ml/Kg of CCl4 dissolved in olive oil will be given to the rat model once/week for 6 consecutive weeks to promote the carcinogenic effect of DEN. Rats with HCC will have a high level of the serum tumor marker alpha-fetoprotein (AFP). After 13 weeks of induction, the animals were assigned into two main groups (control and HCC groups) and then the HCC group subdivided into three groups as follows; Six healthy male rats from group I (the control group) was given saline solution as usual and olive oil as a vehicle. Twenty four male rats made up group II (HCC group), which further got separated into the following four subgroups. Six HCC rats were in group IIA (the untreated HCC group), which got nothing except sterile normal saline and olive oil as a carrier, group IIB (ATX-treated group): consisted of 6 HCC rats that treated with ATX dissolved in olive oil at a dose of 25 milligrammes per kilogramme administered orally five days per week for four weeks; group IIC (DOX-treated group): consisted of 6 HCC rats that treated with DOX dissolved in normal saline at a dose of 2 mg/kg/week via intraperitoneal administration, group IID (Combination-treated group): Consisted of 6 HCC rats that treated with ATX dissolved in olive oil (5 days a week, oral gavage at 25 milligrammes per kilogramme) and DOX dissolved in normal saline (2 mg/kg weekly, intraperitoneal).
At the end of the specified treatment period, rats in all studied groups were sacrificed by deep anesthesia. Then blood samples were collected for assessment of serum ALT, AST, and AFP with MDA determination. Then, liver tissue was dissected out for the process of making tissue homogenates for Nfe2l2, MDA, and glutathione determination, and for the quantitative real-time polymerase chain reaction (qPCR) to measure gene expression, total RNA extraction for expression of PGC-1 α, TFAM, MFN2, Drp1, PINK1 and DNA extraction is required for evaluation of hepatic mtDNA-CN and 8-OHdG. The other part of excised tissue was used for histopathological observation.
The results of the current study indicated that the chemically- induced HCC carcinogenesis was associated with disturbed liver tissues expression of genes controlling the oxidative stress, mitophagy, mitochondrial dysfunction, and antioxidant pathways.
After treatment with ATX alone or combined with DOX, rats presented significant suppression of Drp1, PINK1, and 8-OHdG expression levels with significant upregulation of PGC-1α, TFAM, MFN2 expression levels, a significant improvement of oxidative stress biomarker and marked changes in liver histological structures, liver enzymes tests, and alpha fetoprotein levels. Owing to its synergistic effects of combination treatment with DOX, together with its safety make ATX a novel therapeutic agent for HCC.
from this study, we concluded that
• The used HCC-rat model showed marked shift in the mitochondrial homeostasis in the hepatic tissues manifested as impairment of mitochondrial biogenesis and fusion and induced mitochondrial fission and mitophagy. These abnormalities are caused by marked impairment of glutathione system and induced oxidative stress and DNA damage.
• Astaxanthin targets multiple signaling pathways that play key roles in the development and progression of HCC.
• The treatment with Astaxanthin alone or combined with DOX increased hepatic antioxidants, reduced oxidative stress markers and minimized cytotoxicity of DOX on normal tissue without interfering with DOX cytotoxic effects on tumor tissue.
• Astaxanthin has anti-cancer effects by stimulating the mitochondrial biogenesis pathway in a chemically-induced HCC rat model through modulation of the expressions of master regulator; PGC-1α and TFAM.
• Astaxanthin significantly modulates the mitochondrial homeostasis by inducing mitochondrial fusion and suppressing mitochondrial fission and mitophagy.
• All of these effects of ATX make it a promising therapeutic agent for HCC management.
Further research with a wider range of ATX concentrations and more time intervals on animal models are necessary as ATX may be a promising treatment strategy for HCC.