| Aim:The study was designed to evaluate the decrease-ALT activity and mechanism of huganpian against carbon tetrachloride induced hepatotoxicity in rats.Methods:Male Wistar rats were divided into three groups.Group I served as normal control. Group Ⅱ served as intoxicated control group.GroupIII received the prepared suspension at a dose level of1700mg/Kg b.w.once daily.The animals were treated for9days and on the9th day after one hour of dosing,the toxicant CCL4solution(0.2ml/100g i.p.)was administrated to all the group except Group Ⅰ.After24hours,the blood and livers of animals were collected for the assessment of parameters.The ALT activity of serum and liver was assessed by the colorimetry.Western blotting was used to assess the content of the ALT-1、ALT-2;The expressions of ALT mRNA was measured by RT-PCR; Fluorescence spectrophotometer was used to assess the mitochondrial membrane potential, mitochondrial free Ca2+concentration and cytosolic free Ca2+concentration. Ultraviolet-visible spectrophotometer was used to assess mitochondrial ATPase activity and the sensitivity of mitochondrial swelling.MPTP was measured by ELISA.Results:1. Results of Huganpian efficacy research:There was significant rise in ALT in serum and hepatocytes in CCl4intoxicated control group.Treatment with huganpian prevented rise in ALT in serum and in liver in treated group.The expressions of ALT1and ALT2protein in liver increased in control group Ⅱ and treated group. ALT-1protein was1.47-fold increased and ALT2protein1.83-fold increased relative to their expressions in normal control group mice. ALT-1protein was1.42-fold increased and ALT2protein1.49-fold increased relative to their expressions in normal control group mice.The results showed a correlation between ATL activity in serum and liver tissue protein expressions for both ALT1and ALT2.2. Results of RT-PCR:Conversely, liver ALT mRNA expressions in group Ⅱ and huganpian treated group were decreased. In the group Ⅱ, ALT1mRNA0.42-fold and ALT2mRNA0.48-fold compared with normal control group. In the huganpian treated group, ALT1mRNA0.52-fold and ALT2mRNA0.47-fold.3. Results of mitochondrial function and cytosolic free Ca2+concentration.Mitochondrial membrane potential levels were decreased in group Ⅱ while groupⅢ showed significant rise.The sensitivity of mitochondrial swelling induced by high concentration Ca2+is decreased in group Ⅱ while groupⅢ showed significant rise. Mitochondrial Na+-K+ATPase and Ca2+-Mg2+ATPase activity were rise in group Ⅱ while groupⅢ showed significant decrease.The activity of MPTP was rise in group Ⅱ while groupⅢ showed significant decrease. Mitochondrial free Ca2+concentration and cytosolic free Ca2+levels were rise in group Ⅱ while groupⅢ showed significant decrease.4.Results of serum ALT metabolism;The activity of ALT in serum increased between0-84h in normal control group.The serum ALT activity in CCl4intoxicated control group and treated group decreased between0-84h.The t1/2of serum ALT activity in treated group mice is between72-84h.The t1/2of serum ALT activity in control group mice is longer than84h.These findings suggest that the eliminate rate of serum ALT activity in treated group mice is faster than that of CC14intoxicated control group.Conclusion:1. Treatment with huganpian prevented ALT activity rise in serum and in live.The expressions of ALT1and ALT2protein in liver increased in control group Ⅱ is the base of activity rise.Huganpian can prevent the rise of ALT1and ALT2protein.2. Liver ALT mRNA expressions in control group Ⅱ were decreased. While the liver ALT-1mRNA expressions in huganpian treated group is recovered.3. Huganpian could decrease the release of ALT through protecting mitochondria and decrease the cytosolic free Ca2+level.4.Huganpian could promote the metabolic rate of serum ALT. |
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