INTRODUCTION:

The liver is the largest solid organ in the body, weighing about 1.5 kg in the adult. Lies in the right upper quadrant of the abdomen and is completely protected by the thoracic rib cage. Completely surrounded by a peritoneal membrane, known as Glisson' bscapsule¹. Its primary function is to control the flow and safety of substances absorbed from the digestive system before distribution of these substances to the systemic circulatory system. Liver is closely linked with the small intestine by processing nutrient enriched venous blood that leaves the digestive tract.

The liver made over 500 metabolic functions, resulting in creation of products that are released into the blood stream (e.g. glucose derived from glycogenesis, plasma proteins, clotting factors and urea), or that are excreted to the intestinal tract bile. Also, several products are stored in liver parenchyma (e.x. glycogen, fat and fat soluble vitamins)². Liver Fibrosis or Hepatic fibrosis is a wound healing response to various chronic stimuli, including hepatitis viral infection, metabolic disorders, alcohol abuse, and autoimmune attacks in liver³. During the course of fibrogenesis, various mediators, which are mainly produced by Kupffer cells, resident hepatic cells, and infiltrating inflammatory cells, activate my fibroblasts, causing excess extracellular matrix (ECM) accumulation. Fibrosis is the resulting imbalance between ECM production and resolution. The excessive ECM deposition (especially type 1 collagen deposition) disorders the normal architecture of the liver, resulting in fibrosis progression and subsequent cirrhosis ⁴. Primary biliary cirrhosis, is an autoimmune disease of the liver. It results from a slow, progressive destruction of the small bile ducts of the liver, causing bile and other toxins to build up in the liver, a condition called cholestasis. Further slow damage to the liver tissue can lead to scarring, fibrosis, and eventually cirrhosis⁵. Carbon tetrachloride (CCl4) is an organic solvent extensively used in chemical industry. Its hepatotoxicity includes hepatic necrosis and fatty liver⁶ .Moreover, CCl4 has also an ability to inhibit protein production in liver with early hepatotoxic damage⁷. Early studies showed that free radicals, such as oxygen-centered lipid radicals (LO• or LOO, or both), and trichloromethyl (CCl3) are generated during CCl4 metabolism by hepatic cellular cytochrome P450. These radicals can cause hepatic injury resulting from CCl4 exposure. Protein synthesis is a cytoplasmic event in eukaryotic cells, in which mRNA templates in the form of a genetic code are working to direct protein synthesis. A nuclear envelope (NE) of eukaryotic cells causes spatial separation between mRNA and protein synthesis. Thus, mRNA templates in nuclei have to be transported into cytoplasm, a limiting process of protein synthesis. Nuclear membrane associated nucleotide triphosphatase (NTPase), a specific energy driver of mRNA nucleocytoplasmic export, enables mRNA cytoplasmic accumulation,⁸. Our previousmwork demonstrated that reduced NTPase activity in the NE correspondingly decreases mRNA transport. In this report, we studied the role and mechanism of NTPase activity in CCl4-induced inhibition of protein synthesis in liver ⁸.

MATERIALS AND METHODS:

Experimental Animals:

Using 45 adult male rats (Rattus norvegicus) weighting 200-250 gm were obtained from the animals house in high institutes of fertility/University of Nahrain. The animals were housed in the animal house of Faculty of Science, University of Kufa, under standard environment condition (temperature 25-28 C° and 12 hr light-dark cycle) and allowed access to standard laboratory diet and water.

Experimental protocol

The rats were kept in animal house for acclimation to the laboratory condition for two weeks before they were used for the experiment. each group was formed 15 rats:

group (1) rats were administrated of normal saline (as negative control).

group (2) rats administered with CCl₄ for one month

group (3) rats BDL for one week

Blood Collection:

At the end of experiments. Each animal was anaesthetized by the mixture of xylazine 0.1 ml and ketamine 0.5 ml and they were scarified 15. Heart cut was finished with a 5 ml expendable syringe and 2-5 ml blood was drawn delicately and gradually. The blood was put in test tube containing gel and left for 30 minutes in room temperature and used to get serum through centrifugation at 3000 rpm for 15 minutes to separate serum and put in epindroff tubes which kept at (- 20) in a cooler for assurance biochemical examination.

Measurement of Procollagen111 N terminal peptide (PIIINP):

This kit is used to assay the serum Procollagen 3 N terminal peptide (P111NT) in the sample of Rat serum supplied by Bioassay Technology Laboratory, ( Catalog No: E 002 ) .

Hematological Analysis:

The hematological parameters were performed on EDTA blood using Ruby (Abbott., U.S.A). Ruby is hematology analyzers to perform white blood cell (WBC), hemoglobin (HB) and packed cell volume (PCV) on EDTA²⁴. This instrument was used widely in medicine with more than 4000 instruments worldwide. A measurements being made by laser light flow depolarization cytometry. Results are gives through 1 minute on the LCD show, written out on the copier and kept in the resident memory or in an USB key.

Statistical Analysis:

The results were expressed as (Mean ± Standard Error) were performed by using megastat. And all comparison Were performed by Unpaired sample t-test, while the figures constructed using EXEL program. P-value < 0.05 was used as a level of statistically significant ¹³.

RESULTS:

Effect of CCL4 and Bile Duct Ligation on level of Procollagen111 N terminal peptide (PIIINP)

The result in figure (1)when compared male rat’s with control shows significant increase (p<0.05) in serum level of P111NTP

Figure (1): Changes in PNT111P level in male rat’s treated with CCl₄ for one month and BDL for one week.

Values are mean ± SE. *significantly different at p<0.05

Effect of CCl₄ and Bile Duct Ligation on Rats hematological Parameters.:

The result in table (1) when compared male rat’s with control shows significant decrease in Hb level in both of CCl₄ and BDL treatment. Significant decrease in level of PCV when compared with control in both of CCl₄ and BDL treatment. Significant increase in level of WBC when compared with control in both of CCl₄ and BDL treatment.

Table (4-2): Level of changes in hematological Parameters for male rat’s treated with CCl₄ for one month and BDL for one and two weeks.

Groups	Hb (g/dl)	PCV%	*WBC10³**
Control	13.5±0.2	40.5±0.4*	6333.3±210.8*
CCL4	9.6±0.1*	28.8±0.04*	15500±500*
BDL 1	9.5±0.2*	28.5±0.03*	15666.6±210.8*
BDL2	9.2±0.1*	27.6±0.04*	16000±365*

Values are mean ±SE. *significantly different at p<0.05

DISCUSSION:

The results from this study shows a significantly raise in P111NTP (p<0.05) in this groups gives CCl4 and BDL compared with the control, these increased due to serum precollege type III in primary biliary cirrhosis have high range in patients undergo fibrosis and inflammation. A distinct pattern appears to have emerged. precollege type III levels are increased with fibrosis or inflammation. While previous studies have occasionally noted propeptide elevations as well, it should be emphasized that these elevations can never be assumed to originate exclusively from the liver. As patients with fibrotic liver disease may include patients with associated lung or bone disease, serum propeptide elevations may arise from increased collagen turnover in bone (Type I collagen) and lung (Type I or III collagen)⁹. Precollege type III elevations are readily demonstrated in serum. Peptide elevations appear to correlate greatest with underlying inflammation. This study perhaps has shown this more clearly than the previous human studies. In general, clinical liver disease often displays simultaneous variations in fibrosis and inflammation. In the rat CC14 model, fibrosis appears to persist for period of days, although there is a diminution in the degree of inflammation. Furthermore, this study appears to explain why Precollege type III elevations bear a relationship to inflammation at every stage of the CC14 model⁹. PINP propeptides in part are cleared by liver endothelial cells, and these cells may be injured secondary to inflammation due to the BDL, a part of the observed increase could be due to failure of damaged endothelial cells to clear PINP ¹⁰. As an example, patients presenting with alcoholic liver fibrosis with acute inflammation, N terminal propeptide of type III collagen (PIIINP), a marker of type III collagen formation, is significantly increased, whereas in cirrhosis, PIIINP is only moderately increased .This increased level of PIIINP may both be due to impaired clearance but also the inflammation process itself, as collagen type III is present in arteries and synovial that are highly inflamed. As the level of any serological biochemical marker reflects a balance between formation and clearance, this issue deserves further attention¹⁰. Significant decreased in Hb concentration in groups treated by CCl4 and BDL when compare with control this result agree with¹¹. Due to effect of CCl4 on delta amino levalinic acid dehydrogenase (d-ALADS) that is found in RBC this enzyme is necessary for formation process of Hb (Makino et al., 2000), or may be due to effect CCl4 on protophyr in that is principle for formation of Hb^12,13. The result show significant decrease PCV in the groups treated by CCL4 and BDL when compare with control the result agree with¹⁴ .Due to effect of bone marrow and stem cell that responsible for synthesis of red blood cell by ROS generated by CCL4 treatment and bile acid because BDL. The relationship between WBC count and non alcoholic fatty liver disease include (CCL4 and BDL) related with factors including age, gender, BMI, hypertension, hyperglycemia. The results prove that an elevated WBC level is related to NAFLD incidence, a finding which provides novel and powerful evidence for a significant relationship between WBC level and inflammation¹⁵. Cholestasis is present in multiple pathologies including gall stone obstruction of the bile duct, local tumor impingement and drug toxicity^16,17. Persistent cholestasis leads to injury of hepatocytes and bile duct epithelial cells, inflammation, and progression to fibrosis, cirrhosis and death. The predominant hypothesis concerning the mechanism of cholestatic liver injury is the accumulation of hydrophobic bile salts considered to be highly toxic lead to inflammation and increase WBC¹⁸. Neutrophils cause target cell necrosis by generating reactive oxygen¹⁹. BDL triggers progressive recruitment of neutrophils into the liver causing extensive oxidant stress and cell death ²⁰. HMGB1 is a known damage associated molecular pattern (DAMP), which can bind to toll like receptor 4 (TLR4) and the receptor for advanced glycation end products (RAGE) and induce generation of proinflammatory mediators, which can activate and recruit leukocytes to sites of inflammation ²¹. However, the fact that TLR4deficient mice did not show reduced hepatic neutrophil accumulation and injury after BDL did not support a role of HMGB1 or endotoxin in neutrophil recruitment²². In addition, the release of HMGB1 from necrotic cells and the formation of proinflammatory cytokines and chemokines by macrophages in the circulation are inconsistent with the focal bile infarcts caused by neutrophils. It is more likely that chemotactic factors are derived from bile as the extensive pressure building up during bile duct obstruction leads to local ruptures of cholangioles and leakage of bile into the parenchyma²³. Preliminary data from our laboratory have suggested that chemotactic osteopontin fragments in bile can initiate the neutrophilic inflammatory response after BDL ^21.

REFERENCES:

1. Misih, A., Sherif, R.Z., Bloomston, M. (2010). Liver Anatomy. Surgical Clinics of North America, 643–53.

2. Gerard, J. , Tortora., Bryan, H., and Derrickson.( 2008). Principles of Anatomy and Physiology.12^thed, p. 945.

3. Tahan, C. (2010). Melatonin ameliorates liver fibrosis induced by bile-duct ligation in rats. J. Surg .,Vol. 53, No. 5.

4. Friedman, S.L. (2008). Mechanisms of hepatic fibrogenesis. Gastroenterology, 1655–1669

5. Dan, L., Longo, (2012). Harrison's principles of internal medicine. (18th ed.). New York: McGraw-Hill. pp. Chapter 308. Cirrhosis and Its Complications.

6. Masuda, Y. (2006). Learning toxicology from carbon tetrachloride induced hepatotoxicity. Yakugaku Zasshi ., 126: 885-899

7. Quan, J., Yin, X. and Xu, H. (2009). Boschniakia rossica prevents the carbon tetrachloride-induced hepatotoxicity in rat. Exp. Toxicol. Pathol ., Epub ahead of print .

8. Pang, Y.Z. and Tang, C.S.(2003). Phospholipase A2 inhibits nuclear nucleoside triphosphatase activity and mRNA export in isolated nuclei from rat liver. Life Sci.,73: 969-980.

9. Davis, B.H. and Madri, J.A.(1987). An Immunohistochemical and Serum ELISA Study of Type I and III Procollagen Aminopropeptides in Primary Biliary Cirrhosis. American Journal of Pathology, Vol. 128, No. 2.

10. Schytte, S., Hansen, M., Moller, S., Junker, P., Henriksen, J.H., Hillingso, J., Teisner, B. (1999). Hepatic and renal extraction of circulating type I procollagen aminopropeptide in patients with normal liver function and in patients with alcoholic cirrhosis. Scand. J. Clin. Lab. Invest., 59: 627-633.

11. Esraa, M. (2015). Anti hyperlipidemic and Antioidant Activity of Extracts Cinnamomum zeylenicum in male Rats fed a High Fat Diet . A thesis in physiology, Faculty of Science / University of Kufa , Iraq.

12. Tandon, S. H., Singh, S. T., Prassas, S. O., and Siddiqui, M. K. (2002). Reversal of lead induced oxidative stress by chelating agent, antioxidant or their combination in the rat. Environ Res., 90 (1) : 61- 66

13. Suzen, H. L., Duydu, Y. R., Aydin, A. E., Isimer, A. D. and Vural, N. O. (2003). Influence of the delta amino levulinic acid dehydrogenase (d-ALAD) Polymorphism on biomarkers of lead exposure in Turkish storage battery manufacturing workers. Am. J. Ind. Med., 43(2): 162-71.

14. Zainab, S. (2013). Study of the Protective Effects of Ginger Extracts Against hepatic Induced by Carbon Tetrachloride in Rats. A thesis in physiology, Faculty of Science / University of Kufa , Iraq.

15. Lang, E., Gatidis, S., Freise, N.F., Bock, H., Ralf Kubitz, R. and et al.(2015). Conjugated Bilirubin Triggers Anemia by Inducing Erythrocyte Death. Hepatology. Jan; 61(1): 275–284.

16. Kim, W.R., Ludwig, J. and Lindor, K.D.(2000). Variant forms of cholestatic diseases involving small bile ducts in adults. Am. J. Gastroenterol.,95:1130–1138.

17. Poupon, R., Chazouillères, O. and Poupon, R.E.(2000). Chronic cholestatic diseases. J. Hepatol., 32:129–140.

18. Jaeschke, H.(2011). Reactive oxygen and mechanisms of inflammatory liver injury: Present concepts. J. Gastroenterol. Hepatol.,26(1):173–179.

19. Jaeschke, H. (2006). Mechanisms of Liver Injury. II Mechanisms of neutrophilinduced liver cell injury during hepatic ischemia reperfusion and other acute inflammatory conditions. Am. J. Physiol. Gastrointest, Liver Physiol., 2006;290:G1083–G1088.

20. Gujral, J.S., Liu, J., Farhood, A., Hinson, J.A. and Jaeschke, H.(2004). Functional importance of ICAM1 in the mechanism of neutrophilinduced liver injury in bile ductligated mice. Am. J. Physiol. Gastrointest. Liver Physiol., 286:G499–G507.

21. Yang, K., Köck, K., Sedykh, A., Tropsha, A. and Brouwer, K.L. (2013). An updated review on drug induced cholestasis: Mechanisms and investigation of physicochemical properties and pharmacokinetic parameters. J. Pharm. Sci.

22. Allen, K., Jaeschke, H., Copple, B.L.(2011). Bile acids induce inflammatory genes in hepatocytes: a novel mechanism of inflammation during obstructive cholestasis. Am. J. Pathol. 178:175–186

23. Fickert, P., Trauner, M., Fuchsbichler, A., Zollner, G., Wagner, M, Marschall, H.U., Zatloukal, K. and Denk, H. (2005). Oncosis represents the main type of cell death in mouse models of cholestasis. J. Hepatol. 42:378–385.

24. Mathieleers, I., Lsabel, G., Fadila, S., and Rick, G. (2012). Abbott a promise for life. Com, Case book, 1-91.

Received on 08.04.2017 Modified on 21.05.2017

Research J. Pharm. and Tech. 2017; 10(7): 2132-2135.

DOI: 10.5958/0974-360X.2017.00374.2