Background
A complicated chain of events mediated by various inflammatory cells and molecular mediators results in hepatocyte death and a systemic inflammatory response. The duration of the ischemia and the underlying liver disease determine the severity of the inflammatory reaction and organ dysfunction [
1]. Many pharmacological treatments have been discovered to protect the liver from damage. These agents include antioxidants, ozone, adenosine agonists, nitric oxide (NO) donors, sildenafil, and vardenafil [
2]. CCl
4 is a xenobiotic industrial solvent used to cause chemical hepatitis and liver damage in animals. Carbon tetrachloride-induced liver damage is the most commonly used experimental model for assessing a drug’s hepatoprotective efficacy. A single dose of CCl
4, a potent hepatotoxic xenobiotic, causes acute liver necrosis and steatosis [
3,
4].
Mechanistic studies showed that cytochrome P2E1 is a key part of the proposed mode of action because it changes CCl
4 into highly reactive free radical metabolites. Trichloromethyl and trichloromethyl peroxy are very reactive free radicals that can bind to biological macromolecules and help membrane phospholipid fatty acids form covalent bonds. By damaging polyunsaturated fatty acids within cell membranes, free radicals cause lipid peroxidation, resulting in a chain reaction of free radicals [
4]. When the liver fails because of CCL
4, an imbalance between reactive oxygen species and antioxidant defense causes cells to malfunction and liver necrosis [
5]. CCl
4 treatment greatly enhances hepatic enzyme release, cytochrome P450 degradation, lipid peroxidation products, and an inflammatory response [
6]. Its method of action is seen in the liver cytochrome P450 system’s reductive dehalogenation, which produces trichloromethyl free radicals, which quickly combine with molecular oxygen to form trichloromethyl peroxy radicals [
7].
Carbon tetrachloride is a prevalent contaminant in the environment. Workers are particularly vulnerable to high-level exposure through inhalation and skin contact [
8]. On the other hand, the general population may be exposed to low quantities of CCL
4 by atmospheric inhalation [
9]. Among the several methods of administration of CCl
4, the orogastric route is the most commonly employed since it offers significant benefits over other options. Oral administration of CCl
4 in particular needs minimal amounts of CCl
4 and enables direct transport to the liver via the portal vein, reducing extrahepatic effects due to the selective buildup of CCl
4 in the liver [
10].
According to animal welfare science, there is a strong link between animal welfare and animal health conditions [
11]. Since animal welfare discipline is mostly dependent on behavior. As a result, employing animal models to research the therapeutic targets of innovative pharmaceuticals and herbal treatments must be supported by a thorough examination of normal animal behavior using various behavioral assays. This is an important technique in liver illnesses since it provides us with a full description of the many phases of liver problems and therapy, as well as how far they can impact animal behavior [
12].
Tadalafil is a potent and selective phosphodiesterase type-5 (PDE5) inhibitor that was initially studied as a potential antianginal drug but has since grown in popularity in the treatment of erectile dysfunction and pulmonary arterial hypertension. Phosphodiesterases inactivate cyclic guanosine monophosphate (cGMP) to GMP. PDE inhibition enhances and prolongs the cellular responses to NO and its derivatives that cause vasodilation [
13]. It also has clinically approved immunomodulatory action [
14]. It was also utilized as a medical therapy for portal hypertension in patients with compensated cirrhosis, and it may have long-term benefits in compensated cirrhosis cases [
15], cardioprotection in mice [
16], pulmonary hypertension treatment [
17], chronic renal failure prevention [
18], treatment of Alzheimer’s disease, lower urinary tract symptoms caused by nonmalignant prostate hyperplasia, and irritable bowel syndrome [
19‐
21].
Mansour, Salama [
22] study sought to investigate the protective effect of tadalafil, a PDE5 inhibitor, against thioacetamide-induced liver fibrosis. In their study, tadalafil pretreatment protected against thioacetamide-induced liver fibrosis in a dose-dependent manner, as evidenced by the reduction of inflammatory and fibrotic indices. A Bektas, Karakaya [
23] study also found that high doses of tadalafil (10 mg/kg) and pentoxifylline (40 mg/kg) have the best protective effect against ischemia reperfusion-induced liver tissue damage. Furthermore, PDE5 inhibitors have been shown to have a potentially promising role in the treatment of inflammatory processes [
24] as well as anti-fibrotic effects [
25]. Activation of cGMP-dependent protein kinases causes vasodilation, anti-inflammatory, and anti-proliferative effects, as well as a decrease in collagen synthesis [
26‐
28].
Natural medicinal products may hold the key to natural xenobiotic/drug hepatoprotection [
29].
L. sativum is an edible annual herb that grows wild in the Brassicaceae family.
L. sativum is a medicinal plant that originated in Egypt and the Middle East and is now grown all over the world.
L. sativum is used in traditional medicine to treat inflammatory disorders such as diabetes, arthritis, traumatic injuries, and hepatitis [
30].
Various in vitro biological effects of
L. sativum extract have been reported, including antioxidant, anti-inflammatory, antidiarrheal, antimicrobial, antispasmodic, and hepatoprotective action against oxidative damage, and thus have a high potential for use as herbal hepatoprotective or dietary supplements [
30]. Literature on phytochemical studies of
L. sativum revealed the presence of benzyl isothiocyanate, flavonoids, tannins, triterpenes, alkaloids, sterols, and glucosinolates, all of which have antioxidant, anti-inflammatory, analgesic, and hepatoprotective properties [
31‐
33].
According to a study conducted by Al-Asmari, Athar [
34],
L. sativum seeds have enhanced hepatoprotective activity against CCL
4 (1 mL/kg b.w. via the intraperitoneal route)-induced liver failure in rats, which could be attributed to their antioxidant activity combined with the presence of anti-inflammatory compounds in
L. sativum extract. Furthermore, Rajab and Ali [
35] study found that
L. sativum could be used to prevent hepatotoxicity caused by CCL
4 (1 mL/kg b.w. via the intraperitoneal route 2 times weekly for 12 weeks) in rats through anti-oxidant and anti-inflammatory effects. As a result, the purpose of this work is to discover more about the therapeutic potential of tadalafil,
L. sativum, and especially their combinations against new orogastric CCl
4-induced moderate liver damage in rats using biochemical, histological, and immunohistochemical techniques.
Discussion
Liver failure can occur as a consequence of viral contamination, too much drug use, alcohol addiction, and exposure to numerous harmful substances [
53]. The hepatotoxic experimental rat model of CCL
4 damage is physiologically and pathologically remarkably comparable to human hepatotoxic liver injury [
54]. CCL
4 damages the liver by causing oxidative cellular stress, peroxidation of lipid membranes, and inflammation [
55]. The cytochrome P2E1 enzyme in the liver converts CCL
4 to the hazardous reactive trichloromethyl and trichloromethyl peroxide radicals [
56]. These reactive radicals subsequently attach to unsaturated fatty acids in the membranes of hepatocytes, mitochondria, and the endoplasmic reticulum, initiating a chain lipid peroxidation process that causes hepatocyte and intracellular structural damage and death [
57].
Carbon tetrachloride is a potent hepatotoxin that can cause organ problems like liver fatty layer degeneration and centrilobular necrosis [
58]. Its hepatotoxic effect is characterized by increased liver-damaging enzymes and pathological abnormalities. Normalization of these enzymatic parameters indicates improved liver function, while hepatotoxicity is measured by alterations in transaminase and phosphatase levels [
59,
60]. High levels of AST indicate compromised liver function, similar to viral hepatitis, cardiac infarction, and muscular damage. ALT enzymes convert alanine to pyruvate and glutamate, released from hepatocytes into the blood in liver diseases. Elevated ALP indicates liver blockage or illness [
34]. Serum bilirubin levels are elevated due to leakage from hepatocytes to plasma, which is generally caused by hepatic obstruction to bile outflow and cholestasis [
61].
The current study found that both tadalafil and L. sativum had anxiogenic effects when administered alone in CCL4-induced liver failure in rats; however, the combination-treated group appeared to have anxiolytic effects by increasing the length and number of entries in open arms. Furthermore, the number of entries reduced in closed arms for both tadalafil and L. sativum when given alone in CCL4-induced liver failure in rats and reverted to the normal levels in combination-treated rats with decreased time spent.
The elevated plus maze is a classic rat behavioral test that has been validated for studying the anti-anxiety effects of pharmacological medications [
62]. Anxiolytic drugs preferentially enhance exploration of the open arms while decreasing exploration of the enclosed arms, and anxiogenic drugs selectively reduce exploration of the open arms while increasing exploration of the closed arms. Also, in the elevated plus maze anxiety test, there is a growing notion that NO may cause anxiety [
63]. The NO-cGMP pathway is well known to regulate anxiety in rats. However, research is mixed as to whether stimulating the NO-cGMP pathway increases or decreases anxiety-like behavior.
The bulk of research indicates that inhibiting the NO-cGMP pathway is anxiolytic and activating it is anxiogenic [
64]. Chronic sildenafil or tadalafil administration has antidepressant-like effects in rats, but only when combined with muscarinic receptor antagonism [
65]. According to Balgoon [
66],
L. sativum therapy in Alzheimer’s disease-induced rats reduced elapsed time significantly, indicating better memory and learning. The memory and learning benefits of LS shown in this study might be attributed to lower acetylcholinesterase activity, which improves cholinergic neurotransmission.
The current study found that
L. sativum and tadalafil, particularly the combination group, normalized high levels of AST, ALT, ALP, and bilirubin and decreased total protein activity in an orogastric CCL
4-induced liver failure rat model. Similarly, according to Al-Asmari, Athar [
34] research, pretreatment with
L. sativum seeds and silymarin reduces liver damage induced by intraperitoneally injected CCL
4 by lowering AST, ALT, ALP, and bilirubin levels. In addition, Rajab and Ali [
35] study found that a 12-week pretreatment with
L. sativum extract effectively reduced liver damage induced by intraperitoneal CCL
4 injection by reducing blood liver enzymes and inflammatory biomarkers. Both of the previous investigations looked at the preventive impact of
L. sativum against CCL
4-induced liver damage, whereas the current study looked at the potential therapeutic effectiveness.
Tadalafil’s activation of cGMP-dependent protein kinases causes vasodilation, anti-inflammatory, and anti-proliferative effects, as well as a decrease in collagen formation [
18,
67]. PDE5 inhibitors have been found to have anti-fibrotic properties as well as a potentially promising function in the treatment of inflammatory diseases [
25]. Similarly to our present investigation, Broermann, Schmid [
25] discovered that anti-fibrotic effects caused by the PDE5 inhibitor are represented by differently expressed miRNAs in the liver and reduce CCL
4-induced chronic liver failure in rats. Furthermore, fibrosis generated by thioacetamide injection twice weekly for 6 weeks in rats was mitigated by tadalafil pretreatment via stabilization of inflammatory and fibrotic biomarkers [
22]. However, in the current investigation, we demonstrated the therapeutic benefit of tadalafil in rats with acute liver failure caused by CCL
4, particularly when combined with
L. sativum extract, which showed a more significant therapeutic effect.
The oxidative stress caused by CCL
4 deactivates cellular anti-oxidative enzymes, including peroxidase, catalase, and superoxide dismutase, which neutralize free radicals [
60]. This leads to a buildup of O
2− and H
2O
2, causing liver damage. Lipid membranes are exposed to oxidative stress due to high levels of polyunsaturated fatty acids and transition metallic elements, which can damage cellular proteins, DNA, and inhibit antioxidant enzymes and degrade lipid membranes via the oxidative Haber-Weiss reaction [
68].
The study found that CCL4 poisoning led to a significant increase in MDA levels and decreased antioxidant enzymes SOD and GPx activity. These alterations returned to normal after treatment with
L. sativum and tadalafil, especially in combination. These findings are consistent with the findings of [
22,
35]. Up-and-down inflammatory disorders accompany liver dysfunction/failure. TNF-α, a pro-inflammatory mediator, contributes to oxidative stress-induced liver damage, leading to apoptotic cell death and fibrosis. Kupffer cells release cytokines, chemokines, and pro-inflammatory mediators, initiating hepatic inflammation [
60,
69]. In this study, CCL
4 poisoning increased TNF-α and IL-1β levels. Treatment with
L. sativum and tadalafil, in combination, reverses these alterations, demonstrating potent anti-inflammatory activity.
According to Toriumi, Horikoshi [
70] research, diacylglycerol-O-(OH) is produced during the CCL
4-induced liver damage process, culminating in activation of the protein kinase C/ NF-κB pathway and TNF-α mediated exacerbation of liver injury. Immunohistochemistry staining for NF-kB was significantly greater in the CCL
4 treatment group compared to the CCL
4 and
L. sativum and CCL
4 + tadalafil treatment groups in the current study. The CCL
4 +
L. sativum + tadalafil-treated group and control groups did not exhibit any expression. The NF-κB pathway is involved in the regulation of inflammatory responses as well as the control of apoptosis [
71,
72].
Hepatotoxicity induced by CCL
4 resulted in vascular and hepatic modifications such as thrombosis, congestion, vacuolar degeneration, and mononuclear cell infiltration in the current investigation. CCl
4-induced liver damage is commonly used to evaluate hepatoprotective medicines. Hepatotoxicity is caused by the biotransformation of CCL
4 into free radicals [
73]. The liver, according to Veidal, Karsdal [
74], is a target organ for CCL
4 toxicity due to its function in the body’s defense through detoxification. Because CCL
4 is a well-known hepatotoxic commercial solvent, it is used in a number of experimental models. In contrast to these minor hepatic modifications, subcutaneous injection of CCL
4 (2 ml/kg b.w.) induced obvious hepatic necrosis, inflammation, fatty change, and fibrosis in rats after 12 weeks [
75]. Furthermore, Hassanein, Al-Emam [
76] noted that there were no major histological abnormalities in our investigation, including centrilobular necrosis and visible fatty alterations, due to the oral route and short period of CCL
4 administration. With vacuolar disintegration of the hepatocytes, the liver tissues improved in the CCL
4 and
L. sativum-treated groups. CCL
4 and tadalafil caused vacuolar degeneration of hepatocytes as well as central vein congestion. CCL
4,
L. sativum, and tadalafil treatment groups enhanced the appearance of liver cells. These findings are consistent with those of [
22,
34].
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