exploring hepatoprotective agents and models

The liver, the body’s largest internal organ, plays vital roles in metabolism, detoxification, bile production, and nutrient storage. Liver toxicity can arise from alcohol, chemicals, infections, autoimmune disorders, cancers, and especially drug-induced liver injury (DILI). Common hepatotoxic drugs include anti-tubercular agents, NSAIDs, anticonvulsants, antimicrobials, and anesthetics.

Hepatoprotective agents (e.g., ursodeoxycholic acid, silibinin, N-acetylcysteine, SAMe, silymarin, antioxidants, vitamins) protect against liver injury by reducing oxidative stress, fibrosis, and necrosis.

Evaluation of hepatoprotective activity is done through:

* In vitro models: hepatocyte cultures, stellate cell cultures, collagen stability assays.
* In vivo models: animal studies using liver injury induction (e.g., carbon tetrachloride, galactosamine, allyl alcohol, paracetamol, rifampicin + isoniazid).

These models assess biochemical markers, histopathology, and protective effects of drugs against liver injury, providing insight into potential therapeutic applications.

1. Hepatoprotective Activity . . Presented by Guided by Dr. Pallavi Dewangan PG 1 st Year Dept. of dravyaguna MGAC & RC Dr. Jaymala Jadhav Professor Dept. of dravyaguna MGAC & RC

2. Introduction Function of Liver Liver toxicity Drugs causing DILI Hepatoprotective drug classification In-vitro models In-vivo models References Content

3. The liver is the body's largest internal organ that sits on the right-hand side of the belly. The liver is an essential organ that has many functions in the body, including making proteins and blood clotting factors, it breaks down and detoxifies substances in the body and it also acts as a storage unit, manufacturing triglyceride and cholesterol, glycogen synthesis and bile production. Introduction

4. Many different disease processes can occur in the liver, including infections such as hepatitis, cirrhosis (scarring), cancers and damage by medications or toxins. Hepatoprotective agent are those compounds, which mitigate the liver injury caused by hepatotoxic agents thus can prevent damage to the liver. Examples: Ursodeoxycholic acid (UDCA), silibinin , and N-acetyl cysteine (NAC).

5. To explore the classification and evaluation methods of hepatoprotective agents in combating liver toxicity. To analyze in-vitro and in-vivo models used for studying liver diseases and hepatoprotective mechanisms. Aim

6. Function of Liver

7. Liver Toxicity Main causes of liver or hepatic toxicity are as follows: Alcohol consumption (ethanol) Chemicals (carbon tetrachloride, phosphorus, alfatoxin , chlorinated hydrocarbon etc) Drug-Induced Liver Injury Autoimmune disorder Infection (hepatitis A, B, C) Cancer and other growths (Liver cancer, Bile duct cancer, Liver adenoma)

8. Anti tuberculosis drugs Except ethambutol (1st line oral anti-TB drug) all drugs causes hepatotoxicity Anticonvulsant drugs valproic acid NSAIDs Paracetamol , diclofenac , indomethacin , oxicam Antimicrobial drugs Depson , ketoconazole , sulfonamide , anti-retroviral Anesthetics Enflurane , Isoflurane Miscellaneous drug flutamide , statins , labetalol , nicotinic acid, OC pills, propyl-thiouracil Drug causing DILI (drug induce liver injury)

9. N acetylcysteine Penicillamine Anti oxidants Cardiotropin 1 Herbal medications e.g. silymarine S adenosyl methionine (SAM) Vitamins Melatonin Glutathion Beta-carotene List of Hepatoprotective agents

10. In vitro models In vivo methods Primary hepatocytes cell culture Stellate cell culture Kupffer cell culture Liver cirrhosis and necrosis Inhibition of proline hydroxylation Hepatitis in Long Evans Cinnamon Rat Temporary hepatic ischemia Allyl alcohol induced liver necrosis in rat Carbon tetrachloride induced liver fibrosis in rat Galactosamine induced liver necrosis Thiocetamide induced necrosis of liver Paracetamol induced liver damage in rat Rifampicin and isoniazid induced hepatotoxicity in rats Hepatoprotectives drug classification

11. Fresh hepatocyte preparation and primary culture hepatocyte are used. In Vitro models: Primary hepatocytes cell culture

12. The basic method Isolation of hepatocyte by perfusion of liver with collagenase or utilization primary cultured hepatocyte Incubation of the cell culture with hepatotoxin and with or without the test drug. Determination of the viability of the hepatocyte Evaluation parameters Determination of the activity of transaminases released into the medium by the hepatocyte

13. In chronic injury there is stellate cell activation There is secretion of matrix by activated stellate cell results in liver fibrosis and ultimately cirrhosis Stellate cell are isolated from rat liver by collagenase or pronase digestion Test drugs are incubated with activated stellate cell culture by hepatotoxin B. Stellate cell culture

14. Evaluation parameters Morphology of cell, Alpha SMA (S adenosyl methionine ) expression, cell count with thymidine incorporation and inhibition in synthesis of collagen type 1 and 3

15. The thermal stability of the triple helix of collagen is depend on intramolecular hydrogen bond synthesized by the enzyme propyl-4-hydroxylase Procedure Reaction volume of 1 ml contain tris buffer, glutarate ,ferrous oxide, ascorbate , catalase , dithiotreitol inhibitor After incubation at 37 degrees celsius for 30 minutes the generated CO2 is trapped and determined C. Inhibition of proline hydroxylation

16. 1. Purpose and rationale The Long Evans Cinnamon Rat is useful model to study genetically transmitted hepatitis and chronic liver disease Due to excessive copper accumulation in the liver of Long Evans Cinnamon Rat making this animal a model for Wilson’s disease in human (excess copper accumulation in body) In- vivo methods: A. Hepatitis and Long Evans Cinnamon Rat

17. 2. Procedure Long Evans Cinnamon Rat of age of 5 weeks are housed in temperature and humidity controlled room Group of 6 to 10 rats are given different diets on a 15% purified protein diet and supplemented with vitamin or drugs Drugs are supplied via mini pumps intraperitoneal implanted under anesthesia The occurrence of jaundice is easily observable as the time when the ear and tail turn yellow and the urine become bright ending in death of animal within about a week.

18. Hepatocellular function is altered by temporary hepatic ischemia as occurring during surgical management of acute hepatic trauma and being essential during hepatic transplantation. 1. Purpose and rationale Total hepatic ischemia in rats is produced by placing a ligature around the hepatic artery portal vein and common bile duct B . Temporary hepatic ischemia

19. 2. Procedure Male albino rat 300 to 350 g is fasted for 16 hours prior to the experiment but allow water ad libitum The rats are anesthetized lightly with ether and abdomen cavity is opened through a midline incision Portal vein as a hepatic artery and the bile duct are occluded by placing a tourniquets around the vessels

20. Blood pressure is measured by a catheter is inserted into the right femoral artery. During the ischemic period, 0.7ml of saline is given intravenous at 20 minute interval for volume replacement At the end of 60 minute ischemic period the, tourniquet around the portal vein, hepatic artery and the bile duct is removed in order to re stabilize blood flow to the liver The abdominal incision is then closed and the animal receive either saline or the drug

21. 1. Purpose and rationale Administration of allyl alcohol induced liver necrosis in rat which can be partially prevented by treatment with several drugs such as antibiotic . C. Allyl Alcohol induced liver necrosis in rat

22. 2. Procedure Female Wistar rat weighing 120-150 g are fasted overnight with water ad libitum On following morning test drug is given orally or intraperitoneally to 10 rats. After 1 hour 0.4 ml per kg of 1.25% allyl alcohol solution is given orally Test drug given again on second day and sacrifice on 3rd day liver is removed

23. 3. Evaluation parameter The parietal sides of the liver are checked using a stereo microscope with 25 times magnification Focal necrosis is observed as white green or yellowish hemorrhagic area clearly separated from an infected tissue The diameter of the necrotic area is determined using ocular micro meter The value added for each animal to obtain an index of necrosis Mean of necrosis index is calculated and compared with student t test The protective effect is expressed as percentage decrease of the necrosis index versus control

24. 1 . Purpose and rationale chronic administration of tetrachloride to rat induced severe disturbance of hepatic function together with histologically observable liver fibrosis. D. Carbon tetrachloride induced liver fibrosis in rat

25. 2. Procedure Group of 20 female Wistar rat is used weighing 100- 150g The animal are treated orally twice a week with 1 mg per kg carbon tetrachloride dissolved in olive oil 1:1 over a period of 8 week Animal are fed on chow diet with water ad libitum . Control group receive only olive oil. Test and standard drug given twice daily except on Sunday when only one dose is given Animal weighed every week and at the end of 8 week animal sacrifice using and anesthetic ether

26. 3. Evaluation parameter Total bilirubin Total bile acid 7S fragment of type 4 collagen Procollagen 3 N- peptide

27. 1 . Purpose and rationale Single dose or few repeated dose of D galactosamine cause acute hepatic necrosis in rat Prolonged administration lead to cirrhosis E. Galactosamine induced liver necrosis

28. 2. Procedure Divided doses of 100 to 400 mg/ kg D galactosamine are injected to rat IP or IV during day one for induction of liver cirrhosis male Wistar rat weighing 110-180 gm are injected IP three time weekly with 500 mg/ kg D galactosamine over a period of 1 to 3 months Potential protective substance are administered orally with the food every day The rat sacrifice at various time interval and the liver obtained by autopsy

29. 3 . Evaluation The liver are evaluated by light microscope and immuno -histology using antibodies against macrophages, lymphocytes and the extracellular matrix component The extent of liver cell necrosis and Immuno reactivity for macrophages, lymphocytes and extracellular matrix component is graded semi-quantitatively on 0-4 scale 0 - absent 1 - trace 2 - weak 3 - moderate 4 - strong

30. Study Type Model Animal/Cell Type Induction Method Dose Duration Evaluation Parameters In Vitro Primary Hepatocytes Cell Culture Hepatocytes (Rat) Incubation with hepatotoxins (e.g., Carbon Tetrachloride, Galactosamine ) Varies Short-term Cell viability, Transaminase activity Stellate Cell Culture Stellate Cells (Rat) Activation of stellate cells, Proline hydroxylation inhibition Varies Short-term Cell morphology, α- SMA, Collagen synthesis inhibition In Vivo Hepatitis Model Long Evans Cinnamon Rat Genetic model (Wilson's disease) Test drugs supplied via mini-pumps 1 week (until death) Jaundice observation, Survival rate Animal model activity chart:

31. Study Type Model Animal/Cell Type Induction Method Dose Duration Evaluation Parameters Temporary Hepatic Ischemia Male Albino Rat (300-350g) Ligation of hepatic artery, portal vein, bile duct 0.7ml saline, test drug 60 minutes Blood pressure, Liver function tests Allyl Alcohol-Induced Necrosis Female Wistar Rat (120-150g) Oral administration of 1.25% allyl alcohol 0.4ml/kg allyl alcohol, test drug 3 days Liver necrosis, Necrosis index via microscopy Carbon Tetrachloride-Induced Fibrosis Female Wistar Rat (100-150g) Oral administration of CCl4 in olive oil (1:1) 1ml/kg CCl4 8 weeks Bilirubin , Bile acids , Collagen fragments Galactosamine -Induced Necrosis Male Wistar Rat (110-180g) Repeated injection of D- Galactosamine 500mg/kg D- Galactosamine 1-3 months Liver necrosis, Cirrhosis, Immunohistology

32. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4265599/ https://www.spandidos-publications.com/ijmm/42/4/2020 https://www.sciencedirect.com/science/article/abs/pii/S03788741 20336552?via%3Dihub https://pubmed.ncbi.nlm.nih.gov/?term=hepatoprotective+agents https://www.sciencedirect.com/topics/medicine-and- dentistry/liver-toxicity References