Icaritin Alleviate Acute Liver Injury And Its Molecule Mechanism Study

Background and objectiveIcaritin has drawn greater attention in recent years with the antioxidant, prevention and treatment of osteoporosis, cardiovascular disease, neurodegenerative injury protection, prevention and treatment of breast cancer. Icaritin is the separation from Epimedium which is an important traditional Chinese herbal medicine since ancient times. Icaritin can be obtained by the multi-step extraction and silica gel chromatography, eluting from the precursor named icariin.Icaritin and its precursor icariin have very similar molecular structure. The difference lies in the A ring of the 7-subunit chain has been replaced by–OH(Figure 1). Structure-activity relationship of drugs from the perspective of the theory that the antioxidant function and estrogen receptor-like role will be enhanced. Icaritin current research has confirmed: (1) by reducing PC-3 cells the mitochondrial membrane potential to reduce pRb, Cyclin D1 and CDK4 protein phosphorylation system, the resistance of prostate cancer cells in G1 phase and thus inhibit cancer cells Proliferation; (2) In the primary culture of nerve cells in pharmacological research found that it could be adopted by the estrogen-like effects caused by cell proliferation, reducingβ-amyloid damage to the nerve cells; (3) It can induce differentiation of embryonic stem cells into cardiac muscle cell. (4) It can reduce the hypoxic injury by inhibiting the expression of TPK in the vascular endothelial cells; (5) By protecting the gap protein between the cell it can alleviate the damage of vascular endothelial cells induced by hypoxic injury; (6) It may promote the proliferation of osteoblast cells, inhibit osteoclasts, thus prevent and treat osteoporosis through its antioxidant effect. A variety of reasons leading to liver injury is frequently encountered in clinical work. The protection of the liver has been a hot issue. Related to the protection of liver injury in drug development, gene therapy, surgical intervention has been more active in the field, but still lacking the highly efficient multi-target drugs. Liver damage in the pathological process, the reactive oxygen species cluster of cells lipid, protein, nucleic acid of the total damage is the role of the way. Reducing the lipid peroxidation damage, increasing blood flow to alleviate the acute liver injury related to estrogen receptor by is more of the reports. With the research background in our lab, it can alleviate the ischemia-reperfusion injury of the fatty liver significantly in the mouse model. We can suppose that it might alleviate the acute chemical-induced liver injury. It is expected to become the effective drug in the treatment of acute liver injury. This paper focus on the the function of icaritin which chelate reactive oxygen species, mimic th estrogen-like effect in vitro and in vivo experiments of the acute liver injury. The effect of icaritin in the initial treatment of acute liver injury was evaluated and its possible molecular mechanism was explored.Methods1. Icaritin chelate the reactive oxygen species, and reduce the liver lipid peroxidation damage of primary hepatocyte in vitro.The CCL4 injury model of primary rat hepatocytes was established. Icariin was applied as the positive control for drugs in different doses of Icaritin. The chang of the cells viability was detected by MTT method. The culture supernatant ALT, AST levels was determined by automatic biochemical analyzer. Determination of cell culture supernatant MDA, intracellular SOD was done by absorption spectrometry. Fluorescence microscope detected the strength of reactive oxygen species after pretreated by Icaritin.2. Icaritin increase L-FABP levels through mimicking the role of estrogen-like cells with, so as to decreas the apoptosis by increasing BCL-2/BAX of the liver cell in vitro.The apoptosis model of primary rat hepatocytes induced by CCL4 injury was established. ICI182,780 had been adopted as the estrogen receptor blocker drugs, western blot method was applied to test the different concentrations of PPARαand L-FABP expression at a different time or at different concentration. BCL-2/BAX protein ratio was analyzed by Western blot on the liver cell injury after pretreatment by icaritin.Staining methods were used to detect L-FABP expression level, and flow cytometry method was used to analyze the the proportion of liver cell apoptosis and morphological changes of apoptosis of the liver cell was oberserved by TUNNEL.3. The effect of icaritin on the treatment of acute liver injury in experimental animal models.The acute liver injury model of C57BL/6 female mice induced by CCL4 was established. ICI182, 780 was adopted as the estrogen receptor blocker drug. The use of the spectrophotometer was used to measure serum ALT, AST levels and liver tissue MDA, SOD. The PPARαand L-FABP level of transcription in the liver tissure after pretreatment with icaritin were surveyed by Realtime-PCR method. HE was used to assess the degree of liver injury in the different group. The immune staining methods were used to detect L-FABP expression of the liver tissure. TUNNEL method was used to detect the propotion of apoptosis cell and the reciprocal morphological changes were observed simutinously.ResultsIn vitro study:1. Icaritin increases the survival rate of the cell injured by CCL4.With the increasing of concentration of CCL4 the primary rat hepatocytes survival rate decreased in a dose-dependent manner. icaritin can convert the chang. However, when the concentration rose to 100μmol/L cells survival rate was no longer increasing, showing a downward trend. At the same concentration icaritin improved the survival rate of hepatocyte as twice much as Icariin did.2. The impact of Icaritin on the ALT and AST in the culture medium of primary hepatocyte injuried by CCL4.Icaritin can reduce the release of ALT and AST, with a dose-dependent manner in the scope of 0.1uM-10uM. The effect of 0.1uM, 1uM, 100uM icaritin groups were better than the model group (P<0.05), and the 10uM Icaritin group was the best (P<0.01). The 10uM icaritin was two times better than the same concentration of Icariin.3. The impact of Icaritin on the MDA, SOD and ROS of primary hepatocyte injuried by CCL4.Icaritin can reduce the content of MDA significantly and recover the activity of SOD in a dose-dependent manner. Icaritin can also reduce the generation of reactive oxygen species cluster. The fluorescent light of the 10uM Icaritin group was lower than the the 10uM Icariin group.4. The impact of Icaritin on the PPAR-αand L-FABP in primary hepatocyte injuried by CCL4.The icaritin can upregulate the protein express of PPAR-αand L-FABP in the role of primary rat hepatocytes after PPAR-α, L-FABP in a concentration and time-dependent manner. The results of Western-blot and immunohistochemistry have all shown that the PPAR-αand L-FABP have expressed at a certain level of expression in control group, and decreased after the injury induced by CCL4. The 1uM icaritin group had the remarkable expression than the 1uMIcaritin+ICI 182,780 group (P<0.01).5. The impact of Icaritin on the express of Bcl-2/Bax protein and the apoptosis ratio of hepatocyte injuried by CCL4.The expression of Bcl-2/Bax in the model and vehicle group was decreased than the control group. The different icaritin can increase the ratio of Bcl-2/Bax expression in a dose-dependent manner. But the effect can be blocked by ICI182,780, which was a non selective estrogen receptor blocker.after the block, Bcl-2/Bax ratio has declined markedly. The results of TUNNEL and flow cytometry showed that apoptosis rate of the 1uM Icaritin was significantly lower than the model group and vehicle group (P <0.01). The rate of apoptosis of 1uM icaritin group are also lower than the ICI182,780 group (P <0.05).In vivo study1. The impact of Icaritin on the ALT and AST of the serum of mice with acute liver injury.The level of ALT, AST of the 1mg/Kg icaritin group was lower than model, vehicle and Estrogen group (P<0.05). The level of ALT, AST of the 1mg/Kg icaritin+ICI182,780 group was higher than the 1mg/Kg icaritin group (P<0.01).2. The impact of Icaritin on the MDA and SOD of the liver tissue homogenate of mice with acute liver injury.The level of SOD of the 1mg/Kg icaritin group was higher than model, vehicle and Estrogen group (P<0.05). The level of SOD of the 1mg/Kg icaritin+ICI182,780 group was lower than the 1mg/Kg icaritin group (P<0.05). The variation of MDA was adversely changing compared with the changing tendency of SOD (P<0.05).3. The impact of Icaritin on the PPARαand L-FABPmRNA transcription levels of the liver tissue homogenate of mice with acute liver injury.The PPARαtranscription level was not changed with the control group, but the L-FABPmRNA was a little decreased. The PPARαand L-FABPmRNA transcription level was increased remarkalely in the 1mg/Kg icaritin and E2 group (P<0.01).The PPARαand L-FABPmRNA transcription level was decreased remarkalely in the ICI182,780 group compared with 1mg/Kg icaritin and E2 group (P<0.01).The immunohistochemical staining showed that the expression of L-FABP in the model vehicle group was lower than and control group. The L-FABP level was upregulated remarkalely in the 1mg/Kg icaritin and E2 group (P<0.01).The L-FABP level was downregulated remarkalely in the ICI182,780 group compared with 1mg/Kg icaritin and E2 group (P<0.01). 4. The impact of Icaritin on the degree of the liver tissue injury of mice induced by CCL4.The liver damage points of the model and vehicle group were 3. The liver damage points of the 1mg/Kg icaritin and E2 group were 0.8 and 1.2 respectively, which were significantly severer than the model and vehicle group (P <0.01). The liver damage points of the 1mg/Kg icaritin+ICI182,780 group was 1.4, which were significantly severer than the 1mg/Kg icaritin group (P <0.05).5. The impact of Icaritin on the apoptosis index of of the liver tissue of mice induced by CCL4.The apoptosis index of the 1mg/Kg Icaritin group was lower than model, vehicle and Estrogen group (P<0.01). The level of apoptosis index of the 1mg/KgIcaritin+ICI182,780 group was higher than the 1mg/Kg Icaritin and E2 group (P<0.05).Conclusions1. As a member of Flavonoids, Icaritin strongly chelated ROS, which can allevate the hepatocyte injury induced by peroxidation.2. Icaritin can upregulate the expression of L-FABP protein of hepatocyte through the estrogen receptor signal pathway in a gene manipulated manner which play a important role in the antioxidant function.3. Icaritin can reduce the apoptosis ratio of hepatocyte induced by CCL4 through the manipulating the expression of Bcl-2/Bax.4. Icaritin is an effective multi-target monomer drug to reduce the acute liver injury.In short, we have established the model of acute liver injury induced by CCL4 with the primary rat hepatocytes and mice. The function and mechanism of icaritin alleviating the acute liver injury have been inveatigated by applicating a variety of experiment methods. The results verified the viability of icaritin in allevating the oxidative injury and progression of apoptosis of hepatocyte. It will provide the potent supporting evidence to apply the new drug in clinic.