| Iron is an indispensable element of all living organisms,it is the composition of hemoglobin,myoglobin,cytochrome and some respiratory enzymes(such as cytochrome,cytochrome oxidase,catalase,etc.),it involves in transport and exchange of oxygen and carbon dioxide and promote the transport of lipids in the blood.The iron metabolism of the normal body maintains at steady level and plays an important biological function.Hepcidin is the most important regulator of iron metabolism in the body.It is mainly secreted by the liver in vivo,and its biological function is achieved by interaction with its receptor protein Ferroportin.The hepcidin-ferroportin axis is fundamentally responsible for regulating the supply,utilization,recovery and storage of iron.Hepcidin gene specifically expresses in the liver.The lack of hepcidin which enhances iron uptake and macrophage release in the diet leads to excessive amounts of iron in the serum and organs.Excessive amounts of iron is closely related to the morbidity of these organs.Hereditary hemochromatosis mostly dues to HFE gene mutation,the patient appears liver hepcidin expression decreased,causing abnormal body iron overload.Thus increasing liver hepcidin expression can be used as a method of treating iron overload disease.In the preliminary study of the study group,we used the iron metabolic cell model to screen out a number of natural compounds that affect the expression of hepcidin gene.The effect of berberine hydrochloride on the expression of hepcidin was demonstrated in vitro.On the basis of this,we further find that in Hepal-6 cells,berberine hydrochloride directly promotes the expression of hepcidin by increasing phosphorylation level of Smadl/5/8 and the increase of ERK phosphorylation and decrease of TMPRSS6 expression levels influence the level of phosphorylation of Smadl/5/8,and then regulate hepcidin levels in Hepa1-6 cells.In vivo experiments,berberine hydrochloride could increase the expression of hepcidin in liver of BALB/c mice and change the iron balance in mice under physiological and pathological condition.Berberine hydrochloride has no effect on the iron balance of the hepcidin knockout mouse model,and it illustrates that its main target is hepcidin in the liver.The effects of berberine hydrochloride on the expression of serum hepcidin,serum iron,liver iron and spleen iron in mice were observed after prolonged exposure to berberine hydrochloride at 4 weeks old Hfe-/-mice.It illustrates therapeutic action of berberine hydrochloride on hereditary hemochromatosis disease mouse model.In the above studies,it was confirmed that berberine hydrochloride could increase the expression of hepcidin in vivo and in vitro.The pathway of hepcidin expression in vitro was explained.In vivo,we find the regulating action of berberine hydrochloride on the mouse liver and the influence on the iron balance of the body.Meanwhile it showes a certain therapeutic effect on the Hfe-/-model.Therefore,berberine hydrochloride has shown great potential in the treatment of iron metabolism disease,it is worth further study and exploration.The research of this subject includes the following two parts:literature review and experimental research.1 literature review:This part summarize views in the”Advances in Pharmacology of Berberine Hydrochloride".2 Experimental study:including the following five parts.STUDY I:Study on the mechanism of the regulation of hepcidin expression by berberine hydrochloride.OBJECTIVE:To explore the molecular biological basis of the regulation of hepcidin expression by berberine hydrochloride,we conducted a detailed study on the signal pathway involved in Hepcidin expression.METHODS:Western blotting was used.After treatment with berberine hydrochloride to Hepal-6 cells,the cells were harvested in PBS.Total protein was extracted with RIPA lysate(Solarbio,China)and added with protease inhibitor(Roche).SDS-PAGE was analyzed for the same amount of protein lysate for each sample and subjected to Western blot analysis according to standard procedures.The primary antibodies are listed below,Smad 1/5/8 antibody(1:1000;Santa cruze Biotechnology),anti-Smadlantibody(1:1000;Santa cruze Biotechnology),anti-ERK antibody(1:1000;Zhongyu Jinqiao),anti-pERK antibody(1:1000;Zhongyu Jinqiao),anti-Stat3 antibody(1:1000;Cell Signaling Technology),anti-TMPRSS6 antibody(1:2000;Cell Signaling Technology),anti-BMPRIA antibody(1:1000;Cell Signaling Technology)and anti-GAPDH antibody(1:2000;Sigma-Aldrich).RESULTS:At present,it was found that the regulation of hepcidin was affected by multiple signal pathways.However,under normal circumstances,there were two main pathways for the direct regulation of hepcidin expression,Stat3 pathway and Smad 1/5/8 pathway.During the expression of hepcidin,one or both of the two pathways may be activated.Therefore,we detected expression status of signal pathways after the treatment with berberine hydrochloride in mice Hepal-6 cells.We observed that the phosphorylation level of Smad 1/5/8 in Hepal-6 cells was significantly higher than that in the control group at 50μM concentration of berberine hydrochloride,and the expression level was proportional to the time,while Stat3 phosphorylation level was not found obvious change.Thus,we found that berberine hydrochloride increased the expression of hepcidin by activating the Smadl/5/8 pathway in Hepal-6 cells.At the same time,berberine hydrochloride did not change the expression level of Stat3 and Smadl.To further elucidate the expression mechanism of hepcidin,we screened the Channel protein that may affect the expression of Smadl/5/8,and finally found that ERK phosphorylation level was significantly increased compared with the control group,and the expression was proportional to the time.The expression level of TMPRSS6 was significantly decreased compared with the control group,and the expression level was proportional to the time.The BMPR1A expression level did not show significant changes.CONCLUSIONS:In this paper,berberine hydrochloride promoted the expression of hepcidin directly by increasing Smadl/5/8 phosphorylation level,while the increase of phosphorylation of ERK and the decrease of TMPRSS6 expression also affected the phosphorylation of Smad1/5/8 Level,and then regulated Hepal-6 cells hepcidin levels.STUDY II:Study on dynamic Effects of Berberine Hydrochloride on Iron Balance in WT Mice.OBJECTIVE:To confirm the effect of berberine hydrochloride on hepcidin in physiological condition,we used BALB/c mice for further experiments in vivo.METHODS:BALB/c mice were randomized to 9 mice per group(n = 9).Berberine hydrochloride was dissolved in normal saline,and the rats were given oral administration.The dose was 50mg/kg.The mice in the control group were given the same dose of normal saline.For short exposure,the mice were sacrificed at 6 h,12 h,24h,48 h,72 h after administration.For prolonged exposure,mice were sacrificed at 6 days,9 days and 12 days after administration respectively.Serum was isolated for serum iron testing,the mouse liver and spleen specimens were isolated and weighed for the detection of liver and spleen iron,and a small piece of liver specimens was quickly frozen in liquid nitrogen,and then stored in the-80℃ for RNA extraction.RESULTS:The results of animal experiments were consistent with the results of cell experiments.Berberine hydrochloride could increase the expression of hepcidin in liver.The expression of Hepcidin mRNA was increased by about 50%(P<0.05)at 6h after administration,The expression of hepcidin increased by about 2.5 times(P<0.05)compared with the control group at 12 h after administration,and the stimulation of liver hepcidin expression was continuous after prolonged exposure.Serum iron changed with the change of hepcidin in the liver,and the serum iron concentration decreased by about 20%(P<0.05)at 12h after administration,and the serum iron concentration decreased after prolonged exposure at 6d,9d,12d(P<0.05).Compared with the control group,the spleen and iron were significantly increased from 6h to 12 d after administration(P<0.05).At the same time,the effect of berberine hydrochloride on liver iron was not significant(P>0.05),because after receiving external stimulating,the level of liver iron is generally relatively stable,but the spleen,serum iron lead to iron redistribution due to hepcidin expression changes.In addition,short-term and prolonged exposure to mice did not show significant organ toxicity,demonstrating the biological safety of berberine hydrochloride.CONCLUSIONS:In combination with the results,berberine hydrochloride has a strong regulatory effect on the hepcidin level in the liver of WT mice in physiological condition,and then promote the redistribution of iron content in the body.STUDY Ⅲ:Study on the effect of berberine trihydrochloride on iron balance in Hamp1-/-mice.OBJECTIVE:To demonstrate the role of liver hepcidin in the regulation of iron balance,we used Hamp1-/-mice for further experiments in vivo.METHODS:Hamp1-/-mice is a hepcidin knockout mouse model.Hamp1-/-mice do not produce functional hepcidin in the systemic circulation,with severe iron overload in their serum and organs.In order to observe the changes in serum iron more clearly,we gave these mice low iron diet for 2 weeks to exhaust iron.In the case of iron depletion,we can significantly reduce serum iron levels in Hamp1-/-mice.We randomized Hamp1-/-mice to 9 mice in each group(n=9).Berberine hydrochloride was dissolved in normal saline,and the rats were given oral administration.The dose was 50mg/kg.The mice in the control group were given the same dose of normal saline.Hampl1-/-mice were sacrificed at 72h after administration and serum was collected for serum iron detection.Each mouse liver and spleen specimens were isolated and weighed for the detection of liver and spleen.RESULTS:Serum level of iron did not change significantly after exposure to berberine hydrochloride in Hamp1-/-mice,whereas serum level in BALB/c mice decreased significantly at the same concentration and time.In addition,after administration of berberine hydrochloride to Hamp1-/-mice,there was no significant difference in total liver and spleen between the two groups,which was significantly different from that in BALB/c mice.The iron balance has not changed.CONCLUSIONS:Above all,berberine hydrochloride has no effect on the iron balance of the hepcidin knockout mouse model,which directly indicates that the main target of hepcidin is in the liver.Thus,iron metabolism is regulated by the expression of hepcidin in the liver.STUDY IV:Study on the effect of berberine hydrochloride on iron balance in WT mice after EPO injectionOBJECTIVE:To confirm the effect of berberine hydrochloride on hepcidin in pathological condition,we used BALB/c mice injected with EPO for further experiments in vivo.METHODS:BALB/c mice were randomly divided into 3 groups of 9 mice(n = 9).The first group injected with EPO(erythropoietin),the dose of 10U,while given saline irrigation;the second group injected with EPO(erythropoietin),the dose of 10U,while berberine hydrochloride dissolved in physiological Saline,oral gavage,the dose of 50mg/kg;the third group injected with PBS,while given saline irrigation.Mice were sacrificed at 12h after administration.Serum was collected for serum iron detection,a small piece of liver specimens was quickly frozen in liquid nitrogen,and then stored at-80 ℃ for RNA extraction.RESULTS:EPO is the strongest positive regulator of ERFE,which can promote the synthesis of ERFE and inhibit the expression of Hepcidin.Intraperitoneal injection of EPO can cause BALB/c mice liver hepcidin transient decline,while berberine hydrochloride can increase the liver hepcidin expression.The results showed that the first group of mice hepcidin levels decreased by about 70%(P<0.05)compared with the third group,the second group of mice hepcidin levels increased by about 80%compared with the first group(P<0.05).The above results show that berberine hydrochloride can improve the liver hepcidin levels in the pathological state,thereby affecting the iron balance in mice.CONCLUSIONS:In summary,EPO can transient reduce hepcidin levels in liver,while berberine hydrochloride can regulate the liver hepcidin levels in the pathological state.STUDY V:Study on the effect of berberine pentahydrate on iron balance in Hfe-/-mice.OBJECTIVE:To investigate the effect of berberine hydrochloride on iron balance in Hfe-/-mice,and to observe the therapeutic effect of berberine hydrochloride on iron overload in Hfe-/-mice model.We used Hfe-/-mice for further experiments in vivo.METHODS:Hfe-/-mice is a hfe knockout mouse model.Hfe-/-mice model shows iron accumulation in the liver,increased serum transferrin saturation,increased expression of hepcidin,increased iron absorption in the small intestine,and the presence of iron overload in serum and organs,gradually increased with age.We randomized 8-week-old Hfe-/-mice to 9 mice per group(n = 9).Berberine hydrochloride was dissolved in normal saline,and the rats were given oral administration.The dose was 50mg/kg.The mice in the control group were given the same dose of normal saline.Hfe-/-mice were sacrificed at 12h and 24h after administration.Serum was collected for serum iron detection.Each mouse liver and spleen specimens were isolated and weighed for the detection of liver and spleen.Effect of Berberine on iron balance in Hfe-/-mice.After that,we investigated whether berberine hydrochloride can treate with 4-week-old Hfe-/-mice.Four weeks old Hfe-/-mice were randomized to 9 mice per group(n = 9).Berberine hydrochloride was dissolved in saline,and the dose was 25mg/kg.The rats in the control group were given the same dose of normal saline every other day.Hfe-/-mice was sacrificed at 2 weeks after administration.Serum was collected for serum iron and serum hepcidin detection.Each mouse liver and spleen specimens were isolated and weighed for the detection of liver and spleen.RESULTS:Berberine hydrochloride was exposed to 8-week-old Hfe-/-mice,and the serum iron concentration was significantly lower than that of the control group at the two time points(P<0.05),It indicated that berberine hydrochloride can affect the iron balance in 8-week-old Hfe-/－mice.The effect of berberine hydrochloride on the expression of hepcidin in liver was sustained after two weeks of administration in 4-week-old Hfe-/-mice.Serum hepcidin increased by about 2 times after two weeks compared with the control group(P<0.05),and the concentration of spleen iron increased when compared with the control group.Compared with the control group,the iron concentration in the serum was significantly lower than that in the control group(P<0.05).At the same time,the effect of berberine hydrochloride on liver iron was not significant(P>0.05).CONCLUSIONS:Berberine hydrochloride can affect the iron balance of the 8-week-old Hfe-/-mice model.Meanwhile,when berberine hydrochloride exposed to 4-week-old Hfe-/-mice,serum hepcidin,serum iron,liver iron and spleen iron levels were continuous influenced,indicating that berberine hydrochloride has a certain therapeutic effect on this disease model. |
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