Evaluation of kallistatin and some biochemical parameters in rat with experimental liver injury

Kallistatin acts as a negative acute phase protein that reduces expression after lipopolysaccharide-induced inflammation in the liver. In this study, it was aimed to determine whether there is a relationship between the levels of Kallistatin and liver damage in rats with different degrees of liver failure. The material of this study consisted of 32 Wister Albino male rats obtained from Van Yüzüncü Yıl University Experimental Animals Production and Research Center and weighing between 200-400 gr were used. Thirty-two rats were included in 8 rats in each group as to group 1 (control, n=8), group 2 (mild, n=8), group 3 (moderate, n = 8), group 4 (severe, n=8). In order to cause experimental liver damage in rats, carbon tetrachloride (CCl4) solutions of different percentages dissolved in paraffin oil were applied subcutaneously. Group 1: Healthy rats will not be treated with anything other than the normal diet. Rats in this group were named as control (n=8 male rats). Group 2: In this group (n=8 male rats), 25% CCI4 (dissolved in paraffin liquid) solution was applied to each rat at a dose of 2 ml / kg twice a week subcutaneously for 4 weeks. The rats in this group were named as mild group because of mild liver impairment. Group 3: 25% CCI4 (dissolved in paraffin liquid) solution was administered to rats in this group for 4 weeks in 2 ml / kg subcutaneously twice a week (as same as group 2) and the remaining 2 weeks were treated with 2 ml/kg of 50% CCl4 (dissolved in paraffin liquid) solution (for a total of 6 weeks) and the rats in the group were named as the middle group (n=8 male rats) (Table 6). Group 4: the same procedure of group 3 had been applied for this group then proceed with injected subcutaneously of 2 ml/kg of 62.5% CCl4 in paraffin oil treatment for the 2 weeks (on the whole 8 weeks) and this group was named as the severe group (n=8 male rats). Xylazine 60 mg / kg and Ketamine 7.5 mg / kg combinations were administered intramuscularly to rats in group 2 at the 4th week of the study, group 3 at in the sixth week of study, group 4 in the eighth week of the study and control group rats and 20-30 minutes from these applications. Then the rats were sacrificed. Blood samples were taken into anticogulant and non-anticogulant tubes for immediate hematological and biochemical analyzes in the heart of sacrified rats. Hematological parameters, hemoglobin concentration, leukocyte count, platelet count and MCHC values were measured on the same day with the Veterinary Hemogram Device (Veterinary MS4-s-Melet Schloesing Laboratories in France). Blood samples were taken into antiquagulant-free tubes for centrifugation at 3000 rpm for 15 minutes. The sera obtained were stored at - 20 ° C until measurements were made. Serum AST, ALT, LDH and GGT levels were measured by autoanalyser (BS-120 Vet-Mindray) using commercial kits. Serum Rat Kallistatin (Rat Kallistan, Catalog NO: YLA1624RA SERINA4, ELISA kit Ylbiont), Rat collagen typeⅠ(Col I, Catalog NO: YLA0195RA, ELISA kit Ylbiont) and Rat collagen type III (Col III , Catalog NO: YLA0605RA ELISA kit Ylbiont ) levels were measured using commercial ELISA test kits and with the ELISA instrument as specified in the procedure of the test kits. In the statistical analysis of hematological parameters, WBC, Neu, LYM, Mon, PCT levels in liver damage groups were significantly higher than the control group (p<0.05), while MCH and MCHC levels were lower (p<0.05). Although, RBC, Hct and MPV values were found to be mathematically high, and MCV and Hb values were mathematically low, they were not statistically significant (p<0.05). In the statistical analysis of biochemical parameters, in the analysis of serum ALT levels; serum ALT levels of rats with severe liver injury were found to be significantly higher than those of mild and moderate liver injury, whereas ALT levels of mild and moderate liver injury were compared with ALT levels of control group and no significant difference was found. In the analysis of serum AST levels; serum AST levels of rats with severe liver injury were significantly higher than those of rats with mild and moderate liver injury, but there was no significant difference between AST levels of mild liver injury and AST levels of control group. In the analysis of serum GGT levels; Serum GGT levels of rats with severe liver injury were found to be significantly higher than the GGT levels of mild and moderate liver injury rats, whereas there was no significant difference between liver injury groups (mild and moderate) and control group. In the analysis of serum LDH levels; serum LDH levels of rats with severe liver injury; was significantly higher than the LDH levels of rats with mild to moderate liver injury and control group, and also there were no significant difference between mild and moderate in rats belonging to liver injury groups. In the analysis of serum Collagen-I levels; Serum Collagen-I levels of rats with liver injury were significantly higher than those of the control group. Similarly, serum Collagen-III levels of rats with liver injury were significantly higher than those of the control group. The comparison between the groups was statistically exposed a significant difference between the kallistatin levels of mild liver injury and the kallistatin levels of severe liver injury, however there was no difference was found between the kal levels 71 of moderate and severe liver injury groups. Histopathological examination of the liver organ of the liver and control group rats revealed that the liver tissue had normal histological appearance, while the liver tissue of carbontetra chloride (in rats belonging to liver injury groups) showed severe morphological changes. These findings were hydropic degeneration and coagulation necrosis was detected in hepatocytes, especially in the vena centralis and mid-zone regions. Fibrosis was present in some vena centralis (Periacinary fibrosis) and portal areas. The spread of thin collagen bundles from the portal marks to the parenchyma was observed only. Activation was also observed in pericinusoidal myofibroblastic cells. Lymphoid cells mixed with several macrophages were observed to accumulate occasionally in portal scars and liver parenchyma. These changes resulted in cell dissociation and disruption of the liver lobe architecture in the liver parenchyma of heavy CCl4 treatment group rats. When the histopathological examination of the hematological, biochemical parameters and liver tissue of rats with different degrees of liver damage were made with carbontetra chloride, significant changes were found in the control group. The relationship between the severity of liver tissue damage and the Kallistatin levels of the liver tissue of rats with carbontetra chloride (liver injury groups) was demonstrated by both biochemical and hematological parameters. As a result; Changes in the Kallistatin levels of rats with liver injury were found to be significantly low in parallel with the damage to the liver tissue. It was concluded that changes in serum kallistatin levels are an important parameter in determining liver tissue damage levels and evaluating treatment prognosis in the treatment of liver diseases. Key Words: Kallistatin, Liver injury, CCl4, Biochemical parameters