Inhibitory effects of saikosaponin-d on CCl_4-induced hepatic fibrogenesis in rats

AIM: To investigate the suppressive effect of saikosaponin-d (SSd) on hepatic fibrosis in rats induced by CCl4 injections in combination with alcohol and high fat, low protein feeding and its relationship with the expression of nuclear factor-κB (NF-κB), tumor necrosis factor-alpha (TNF-α) and interleukins-6 (IL-6). METHODS: Hepatic fibrosis models were induced by subcutaneous injection of CCl4 at a dosage of 3 mL/kg in rats. At the same time, rats in treatment groups were injected intraperitoneally with SSd at different doses (1.0, 1.5 and 2.0 mg/kg) once daily for 6 wk in combination with CCl4, while the control group received olive oil instead of CCl4. At the end of the experiment, rats were anesthetized and killed (except for 8 rats which died during the experiment; 2 from the model group, 3 in high-dose group, 1 in medium-dose group and 2 in low- dose group). Hematoxylin and eosin (HE) staining and Van Gieson staining were used to examine the changes in liver pathology. The levels of alanine aminotransferase (ALT), triglyeride (TG), albumin (ALB), globulin (GLB), hyaluronic acid (HA) and laminin (LN) in serum and the content of hydroxyproline (HYP) in liver were measured by biochemical examinations and radioimmuneoassay, respectively. In addition, the expression of TNF-α and IL-6 in liver homogenate was evaluated by enzyme- linked immunosorbent assay (ELISA) and the levels of NF-κBp65 and I-κBα in liver tissue were analyzed by Western blotting. RESULTS: Both histological examination and Van Gieson staining demonstrated that SSd could attenuate the area and extent of necrosis and reduce the scores of liver fibrosis. Similarly, the levels of ALT, TG, GLB, HA, andLN in serum, and the contents of HYP, TNF-α and IL-6 in liver were all significantly increased in model group in comparison with those in control group. Whereas, the treatment with SSd markedly reduced all the above parameters compared with the model group, especially in the medium group (ALT: 412 ± 94.5 IU/L vs 113.76 ± 14.91 IU/L, TG: 0.95 ± 0.16 mmol/L vs 0.51 ± 0.06 mmol/L, GLB: 35.62 ± 3.28 g/L vs 24.82 ± 2.73 g/L, HA: 42.15 ± 8.25 ng/mL vs 19.83 ± 3.12 ng/mL, LN: 27.56 ± 4.21 ng/mL vs 13.78 ± 2.57 ng/mL, HYP: 27.32 ± 4.32 μg/mg vs 16.20 ± 3.12 μg/mg, TNF-α: 4.38 ± 0.76 ng/L vs 1.94 ± 0.27 ng/L, IL-6: 28.24 ± 6.37 pg/g vs 12.72 ± 5.26 pg/g, respectively, P < 0.01). SSd also decreased ALB in serum (28.49 ± 4.93 g/L vs 37.51 ± 3.17 g/L, P < 0.05). Moreover, the expression of NF-κB p65 in the liver of treated groups was lower than that in model groups while the expression of I-κBα was higher in treated group than in model group (P < 0.01). The expression of NF-κBp65 and TNF-α had a positive correlation with the level of HA in serum of rats after treatment with CCl4 (r = 0.862, P < 0.01; r = 0.928, P < 0.01, respectively). CONCLUSION: SSd attenuates CCl4-induced hepatic fibrosis in rats, which may be related to its effects of hepato-protective and anti-inflammation properties, the down-regulation of liver TNF-α, IL-6 and NF-κBp65 expression and the increased I-κBα activity in liver.

AIM： To investigate the suppressive effect of saikosaponin-d （SSd） on hepatic fibrosis in rats induced by CCh injections in combination with alcohol and high fat, low protein feeding and its relationship with the expression of nuclear factor-κB （NF-κB）, tumor necrosis factor-alpha （TNF-α） and interleukins-6 （IL-6）. METHODS： Hepatic fibrosis models were induced by subcutaneous injection of CCh at a dosage of 3 mL/kg in rats. At the same time, rats in treatment groups were injected intraperitoneally with SSd at different doses （1.0, 1.5 and 2.0 mg/kg） once daily for 6 wk in combination with CCh, while the control group received olive oil instead of CCh. At the end of the experiment, rats were anesthetized and killed （except for 8 rats which died during the experiment; 2 from the model group, 3 in high-dose group, 1 in medium-dose group and 2 in lowdose group）. Hernatoxylin and eosin （HE） staining and Van Gieson staining were used to examine the changes in liver pathology. The levels of alanine aminotransferase （ALT）, triglyeride （TG）, albumin （ALB）, globulin （GLB）, hyaluronic acid （HA） and larninin （LN） in serum and the content of hydroxyproline （HYP） in liver were measured by biochemical examinations and radioimmuneoassay, respectively. In addition, the expression of TNF-α and IL-6 in liver homogenate was evaluated by enzymelinked immunosorbent assay （ELISA） and the levels of NF-κBp65 and I-κBa in liver tissue were analyzed by Western blotting. RESULTS： Both histological examination and Van Gieson staining demonstrated that SSd could attenuate the area and extent of necrosis and reduce the scores of liver fibrosis. Similarly, the levels of ALT, TG, GLB, HA, and LN in serum, and the contents of HYP, TNF-α and IL-6 in liver were all significantly increased in model group in comparison with those in control group. Whereas, the treatment with SScl markedly reduced all the above parameters compared with the model group, especially in the medium group （ALT： 412 ± 94.5 IU/L vs 113.76 ± 14.91 IU/L, TG： 0.95 ± 0.16 mmol/L vs 0.51 ± 0.06 mmol/L, GLB： 35.62 ± 3.28 g/L vs 24.82 ± 2.73 g/L, HA： 42.15 ± 8.25 ng/mL vs 19.83 ± 3.12 ng/mL, LN： 27.56 ± 4.21 ng/mL vs 13.78 ± 2.57 ng/mL, HYP： 27.32 ± 4.32 ug/mg vs 16.20 ± 3.12 ug/mg, TNF-a： 4.38 ± 0.76 ng/L vs 1.94 ± 0.27 ng/L, IL-6：28.24 ± 6.37 pg/g vs 12.72 ± 5.26 pg/g, respectively, P 〈 0.01）. SSd also decreased ALB in serum （28.49 ± 4.93 g/L vs 37.51 ± 3.17 g/L, P 〈 0.05）. Moreover, the expression of NF-KB p65 in the liver of treated groups was lower than that in model groups while the expression of I-κBa was higher in treated group than in model group （P 〈 0.01）. The expression of NF-κBp65 and TNF-a had a positive correlation with the level of HA in serum of rats after treatment with CCh （r = 0.862, P 〈 0.01; r = 0.928, P 〈 0.01, respectively）. CONCLUSION： SSd attenuates CCh-induced hepatic fibrosis in rats, which may be related to its effects of hepato-protective and anti-inflammation properties, the down-regulation of liver TNF-a, IL-6 and NF-κBp65 expression and the increased I-κBa activity in liver.