MTX is an antimetabolite therapeutics that is extensively used as an anticancer and immune-suppressive drug, although its therapeutic efficacy is hampered by organ toxicities (19). One of the most dangerous side effects of long-term MTX treatment is hepatotoxicity, which is caused by the accumulation of 7-hydroxymethotrexate, the drug's primary metabolite (20). Because of the importance of drug-induced hepatic toxicity in clinical medicine, researchers are increasingly interested in gaining a better understanding of the toxicity, specifically the processes and ways to reduce the occurrence of this nasty side effects. In this study, the ameliorative impacts of morin on MTX-induced hepatocellular toxicities were assessed.
Hepatic dysfunction has been linked to MTX intoxication, as evident by raised serum activity of the ALT, ALP and AST enzymes (21, 22). In this study, serum marker enzymes of hepatocellular injury were evaluated. Under normal circumstances, these enzymes are mostly found in the hepatic tissue. They are frequently released into the circulation for the duration of hepatocyte necrosis or membrane damages. AST and ALT are found in periportal hepatocytes and engage in transamination reactions during amino acid metabolism (23). However, their serum activity have been shown to be enhanced following cellular membrane injury and leaking. As a result, the considerable rise in levels of these enzymes following MTX administration is revealing the disturbed membrane permeability in the treated rats, which is associated with hepatic injury. However, the noteworthy reductions in serum AST, ALT and ALP levels after morin administration to MTX-treated rats suggests that morin provides hepatoprotection against MTX-induced liver injury. In agreement with our findings, morin therapy has been shown to normalize blood biochemical indicators (10, 11).
Hepatic impairment caused by MTX have been linked to oxidative damage. MTX causes oxidative stress at the cellular level by producing free radicals and ROS (24, 25). LPO, alterations in antioxidant machinery, DNA damage, alteration in gene expressions and apoptosis are all symptoms of oxidative stress (26). The organism has evolved numerous mechanisms to defend itself against oxidative stress. These processes include antioxidant enzymes like SOD, CAT, and GPx, as well as the non-enzymatic antioxidant glutathione (27, 28). Several independent studies have been conducted on antioxidants and their importance in preventing oxidative injury and the cellular damage that comes with it, as well as the critical roles of SOD, CAT and GPx (24, 29, 30). The effects of morin supplementation on oxidative stress indicators in MTX-induced hepatotoxicity were studied in this work. The findings demonstrated that MTX administration increased MDA levels and decreased GSH levels as well as SOD, CAT, and GPx activities in hepatic tissues, while morin therapy mitigated these effects. MTX has also been reported to decrease Nrf2 binding activity and suppresses Nrf2 expression in the livers of MTX-treated mice (31). This decrease in Nrf2 activity may be linked to a decrease in the liver's anti-oxidant status. We measured Nrf2-HO-1 pathway mRNA expression to further understand the processes underlying morin's protective impact against MTX-induced liver damage. Nrf2 has been reported to be activated by cytoplasmic Kelch-like ECH-associated protein 1 and stimulates the production of antioxidant genes in response to oxidative stress (32). Nrf2 regulates the transcription of HO-1, an important cellular antioxidant enzyme (33). HO-1 can reduce inflammation, reduce oxidative stress, and slow the rate of apoptosis all at the same time. Colectively, our findings suggest that HO-1 induction through Nrf2 activation may play a role in morin's cytoprotection against MTX-induced oxidative stress.
Apoptosis is a type of biological cellular death that is vital for the formation and maintenance of homeostatic balance in human and animals. Pro-apoptotic proteins like Bax, caspase-3 and anti-apoptotic factors like Bcl-2 strictly regulate it (34). According to the reports, MTX causes liver damage, which may be directly related to pro-apoptotic protein activation. In the current study, we use the quantitative real time PCR method to assess three apoptotic markers: Bax, caspase-3 and Apaf-1 and anti-apoptotic Bcl-2. The mitochondrial apoptosis process is started by Bax, which also damages DNA and activates caspase-3 (35). The intrinsic or mitochondrial mechanism of apoptosis uses the protein Apaf-1, which oligomerizes in response to the release of cytochrome c and creates the huge complex known as an apoptosome. The apoptosome recruits and activates procaspase-9, a mitochondrial pathway initiator caspase, which results in the processing of caspase-3 downstream (36). According to the literature, MTX treatment causes liver apoptosis through effecting modulating levels of Bax, caspase-3, Apaf-1 and Bcl-2 (37, 38). The findings of the current study are consistent with previous findings regarding Bax, caspase-3, Apaf-1 and Bcl-2 regulation. Morin dramatically mitigated Bax, caspase-3, Apaf-1 and Bcl-2 expressions to a level that is comparable to the control.
One of the key elements of the signaling cascade that controls a variety of cellular functions, including cell division, developmental programs, hormone responses, and biotic and abiotic stress responses, is the MAPK pathway (39). The most lately discovered atypical MAPK and one that has received the least attention is called MAPK15. Studies on the function of MAPK15 in many different cells and model organisms show that MAPK15 is involved in a wide range of cellular processes, including stimulating cell division and protein secretion; controlling cell division, cell transformation and apoptosis (40). MAPK14 is a osmoregulatory protein JNK, an important branch of the MAPKs, plays an important function in the apoptosis triggered by TNF-α that is induced via exposure to a variety of cellular stresses (41). One of the biochemical mechanisms leading to the adverse effects of MTX is p38 MAPK-signaling pathway is especially associated with a pulmonary inflammatory response (42). The findings of the current study are consistent with previous findings regarding MAPK signaling regulation (43). Morin dramatically mitigated effects of these to a level that is comparable to the control in parallel to literature (12, 44).
The Akt/PKB isoforms have different roles in animals, with Akt2 primarily regulating metabolic signaling and Akt1 regulating growth and survival (45). FOXO1 is a member of the forkhead transcription factor family, which is involved in a variety of biological functions such as DNA repair, apoptosis, cell cycle arrest, and resistance to oxidative stress (46). The Akt-FoxO1 signaling pathway plays an essential role during liver regeneration (47). The mRNA expression levels of Akt2 and FOXO1 were significantly upregulated in the liver of MTX-induced rats according to our RT-PCR data. Morin (50 and 100 mg/kg) treatment, on the other hand, reduced the levels of these markers. In a different study, morin inhibits glucose production in cultured HepG2 cells inactivating FOXO1 and Akt (48).
Matrix Extracellular matrix proteins are broken down and synthesized more quickly by metalloproteinases (MMPs), a class of zinc-dependent endopeptidases. Gelatinase MMP-2 and MMP-9 are two examples of this category of enzymes. High levels of these gelatinases in the bloodstream are linked to an inflammatory process (49). MMPs have been associated with the pathophysiology of the MTX (50). In current study, we demonstrated that serum MMP-2 and MMP-9 levels increased after MTX administration in consistent with the literature. In contrast to the MTX group, co-supplementation of morin (50 and 100 mg/kg) effectively reversed MTX-induced upregulations in MMP-2 and MMP-9 levels. The impacts of morin on MMP-2 and MMP-9 expressions were investigated in other studies as well (51, 52). In this study, we have demonstrated ability of morin in mitigating MTX-induced hepatic injury.