PROTECTIVE EFFECT OF DIOSGENIN AGAINST CARBON TETRACHLORIDE AND CISPLATIN INDUCED HEPATOTOXICITY IN RATS

Liver fibrosis is one of the most widespread chronic liver diseases in the world. Fibrosis is the wound healing response of an organ and formation of fibrous scar tissue[1].During liver fibrosis there is an excessive production and deposition of extracellular matrix protein (ECM) including collagen which disrupts the normal structure of the liver and cause activation of hepatic stellate cells (HSCs)[2, 3].Nonalcoholic fatty liver diseases (NAFLD), alcoholic liver diseases, chronic hepatitis B (HBV) and hepatitis C (HCV) are the most common causes of liver fibrosis[4, 5].


INTRODUCTION
Liver fibrosis is one of the most widespread chronic liver diseases in the world. Fibrosis is the wound healing response of an organ and formation of fibrous scar tissue [1].During liver fibrosis there is an excessive production and deposition of extracellular matrix protein (ECM) including collagen which disrupts the normal structure of the liver and cause activation of hepatic stellate cells (HSCs) [2,3].Nonalcoholic fatty liver diseases (NAFLD), alcoholic liver diseases, chronic hepatitis B (HBV) and hepatitis C (HCV) are the most common causes of liver fibrosis [4,5].
Fibrosis is the first stage of liver cells scarring and later if more of the liver becomes scarred, it's known as liver cirrhosis. A scar is a fibrous tissue made up of collagen that replaces the injured cells, performs no function, type I and type III are the types of collagen that makeup scar tissue. Typically, a hepatic stellate cells (HSCs) play acentral role in the pathogenesis of hepatic Correspondence: Ravina P Shelke, Department of Pharmacology, Dr. Vithalrao Vikhe Patil Foundation's College of Pharmacy, Vilad Ghat, Ahmednagar (MS), India. Email: rajevikram@gmail.com fibrosis and comprise 15% of the liver cell mass [6]. The activation of Cellular component primarily hepatic stellate cells is important during the initiation and development of liver fibrosis [2]. Hepatic stellate cell stores vitamin A as retinyl esters, due to the liver fibrosis HSCs proliferate, lose their vitamin A and undergo major phenotypical transformation to myofibroblast (activated HSCs) that increasingly express α-smooth muscle actin and produces collagenous extracellular matrix proteins [7].The transforming growth factor-β1 (TGF-β1) is a cytokine, important in hepatic stellate cells activation and proliferation and regulation of ECM formation [8].TGF-β1 is acted in an autocrine and paracrine manner and is produced by kupffer cells [9].
Matrix metalloproteinase (MMP) have capability to degrade the ECM component. But in fibrotic liver there is an expression of tissue inhibitor of metalloproteinases, which inhibit the MMP and play a key role in fibrogenesis. Therefore, the inactivation of activated HSC is a key process for the recovery from liver fibrosis [1]. Fibrosis can occasionally reverse if the cause is identified rapidly. Fibrosis can develop because of continuously damaging cells for month or year. Diosgenin has shown antioxidant, hypolipidaemic activity. This research demonstrated that activity of herbal medicine diosgenin that has acts against liver fibrosis through inhibition of cytokine production [10].

Study design: An experimental animal based study
Ethics approval: The experimental protocol was approved by the institutional animal ethics committee. (COPH/IAEC/PG/05/2018).

Study duration: 28 days
Animals: The healthy Albino Wistar rats of either sex, weighing between 180-250 g were taken for the study. They were housed under standard environmental conditions of temperature (23±20c) humidity (55±5%) and 12h light and 12h dark cycles. The animals were fed with standard pellet diet and water ad labium. The experimental protocol was approved by the institutional animal ethical committee as per the CPCSEA guidelines.

Sample size: In each group n=6
Drug and chemicals: Cisplatin was purchased from Hetero Healthcare Ltd, Diosgenin was purchased from Yarrow Chem Products, Carbon tetrachloride was purchased from Qualigens Fine chemicals and other reagent were purchased from local market, all this chemicals used in this studies were analytical grade.
Dose Selection of Drug: Studies assessing Diosgenin for toxicity study have shown that the compound was usually well tolerated orally in doses of up to 500 mg/kg. The selected doses of Diosgenin 50, 100, 150 mg/kg body weight for the rats. They were given by oral route using oral gavage [11].

Induction of liver fibrosis by Carbon tetrachloride:
The rats were subjected to intraperitoneally administration of 1ml/kg CCl4 mixed with an equal volume of olive oil, twice a week for 28 days.

Methodology
Experimental design: 1. CCl4 Induced liver fibrosis: The animals were divided into 5 groups of six animals in each group.

Cisplatin induced liver fibrosis:
The animals were divided into 5 groups of six animals in each group.

Estimation of different parameters:
After 28 days of both CCl4 and cisplatin induced liver fibrosis methods, the animals were kept to fasting overnight after the experimental period. They were anaesthetized with ether and blood was collected by puncturing retroorbital plexus and left at room temperature for 30 minutes in dry test tubes without any disturbance. Then the blood samples were centrifuged for 10 minutes at 3000 rpm to separate the serum.
Biochemical parameter: At the end of treatment the serum was subjected to various biochemical tests to assess liver function such as aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and total bilirubin (TBL). The livers were homogenized to estimate the level of hydroxyproline (HP).
Morphological parameter: After completion of the experimental period the animals were sacrificed and abdomen was cut and open the liver was isolated and the weight of the liver was measured.
Histo-pathological studies: Livers were dissected out and preserved in 10% formalin for Histopathological studies. Liver sections were stained with haematoxylin and eosin, the stained sections were observed under the microscope to estimate the extent of liver fibrosis.
Statistical analysis: Results were expressed as the Mean ± standard error means (S.E.M). The comparison of data within groups was performed by the analysis of variance using ANOVA test. Significant difference between control and experimental groups was assessed by Dunnetts test. Statistical analysis was performed using INTA. A probability level of less than p<0.05 was considered significant.
Ravine et al.  Protective effect of Diosgenin against Carbon tetrachloride and Cisplatin induced hepatotoxicity in rats.

Effect on CCl4 induced liver fibrosis:
At the end of 28 days treatment of diosgenin in CCl4 induced liver fibrosis, the animals were subjected for biochemical investigation. Serum level of AST, ALT, ALP, TB and HP were analyzed and shown in Table 1.

Effect on liver weight in CCl4 induced liver fibrosis:
When the liver weight of the negative control group was compared with control group, there is significant (##p<0.01) increase in the liver weight of negative control group due to ECM deposition or fatty infiltration. There is significant (** p<0.01) decrease in liver weight of T-50, T-100 and T-150 group when compared with negative control group.

Histopathological examinations
Histopathological examinations of the rat livers in CCl4 induced liver fibrosis:

Effect on Cisplatin induced liver fibrosis:
At the end of 28 days treatment of diosgenin in cisplatin induced liver fibrosis, serum level of AST, ALT, ALP, TB and HP were analyzed. There

Effect on liver weight in Cisplatin induced liver fibrosis:
There is significant (##p<0.01) increase in the liver weight of negative control group (Cisplatin) When com-

Sr.
No. pared with control group. There is significant (** p<0.01) decrease in liver weight of T-50, T-100 and T-150 group when compared with negative control group.

Histopathological examination
Histopathological examination of the rat livers in cisplatin induced liver fibrosis: Figure 6: group-I showed the structure of liver with normal periportal area

DISCUSSION
Many herbal drugs possess hepatoprotective effect hence can be used for the management of various liver diseases. The CCl4 possess hepatotoxic effect in the experimental animals. The enzyme cytochrome P450 biotransformed CCl4 and produce the trichloromethyl free radicals, which cause lipid peroxidation of lipids membrane and peroxidative degradation in the adipose tissue resulting in fatty or ECM infiltration of the hepatocytes. All these events culminate in loss of integrity of cell membrane and damage of hepatic cells [15]. Free radical generation causes oxidative damage due to hepatotoxic effect of CCl4.In hepatotoxicity, cause liver damage due to serum enzymes leak into blood stream. The elevated levels of biomarker (AST, ALT, ALP), TBL and HP are indications of hepatotoxicity in CCl4induced rats.
In our study, a repeated administration of CCl4 for four weeks (twice a week) to the experimental animals led to progressive deterioration of liver structure, with a subsequent liver fibrosis [7]. In the present study, the transaminase (AST, ALT, ALP) level increased by CCl4 treatment was significantly (p<0.01) reduced by diosgenin treatment T-50, T-100 and T150. The total bilirubin and HP level was also restored to the near normal level upon CCl4 intoxication by the diosgenin treat-Ravine et al.  Protective effect of Diosgenin against Carbon tetrachloride and Cisplatin induced hepatotoxicity in rats.
ment. These findings were further supported by histopathological studies, which clearly indicated that the hepatic fibrosis occurred in negative control group and increased liver weight and HP content were also seen in negative control group. HP content is good marker of liver fibrosis. The increased weight of liver by CCl4 was restored by diosgenin (T-150) up to the near normal significantly (p<0.01). This revels that diosgenin treatment prevented the ECM deposition in the liver cells and indicating hepatoprotective and antifibrotic action. The comparative Histopathological study of the liver from different groups of rat corroborated the hepatoprotective efficacy of the diosgenin. Various pathological changes like steatosis, fatty change, areas of severe necrosis, periportal inflammatory infiltrate and periportal fibrosis, associated with liver fibrosis were prevented to moderate extent in treatment group.
High doses of cisplatin have been known to produce hepatotoxicity [16]. Hepatotoxicity induced by cisplatin is recognized by the alteration of the biochemical, histological, and molecular parameters. As ALT exists mainly in the liver cell cytoplasm, it is one of the most sensitive parameters for liver cell function test as recommended by WHO. The result of our study showed that activities of serum enzymes (ALT, AST, and ALP) in negative control group increased in comparison with the control group. ALT, AST, and ALP enzymes levels increase in serum confirmed hepatocytes cell membrane damage and enzymes leak from the hepatocytes [12]. Our result showed that cisplatin makes dosedependent changes in liver structure: some of these changes impair hepatocytes arrangement, liver lobulation, and necrosis. This study also confirmed that addition of some antioxidant foods may have preventive effects on cisplatin induced hepatotoxicity. The difference in weight of animals after the experiment showed a significant difference between normal control and negative control group.
In the present study, administration of Cisplatin for 0, 1, 3 weeks to the experimental animals cause impairment of liver architecture, with a progression of liver fibrosis [7]. In the present study, the transaminase (AST, ALT, ALP) level increased by Cisplatin treatment was significantly (p<0.01) decrease by diosgenin treatment groups. The increased weight of liver by Cisplatin was restored by diosgenin T-150 up to the near normal significantly (p<0.01) after28 days. Result in diosgenin treatment prevented cisplatin induced liver fibrosis. In the Histopathological study of the liver from different groups of rat normal control group shows normal hepatic cells and, where as administration of Cisplatin in negative control group showed portal fibrosis and treated group showed mild ECM infiltration.

CONCLUSION
In conclusion, there have been considerable advances in the understanding of the mechanisms that underlie hepatic fibrogenesis. The central event in fibrogenesis appears to be the activation of HSCs, which is a complex process. Taking into consideration the results obtained in the present investigation, it can be concluded that, Diosgenin treatment exerted significant hepatoprotective effect in laboratory animals by inhibiting ECM deposition and HSCs activation.