Protective Effects Of Salvia Miltiorrhiza On Iron-overloaded Zebra Fish And Inhibitory Effects Of Salvia Miltiorrhiza Against Hepatic And Myocardial Fibrosis In Chronic Iron-overloaded Mice

Iron is an essential micronutrient for living organisms, but it is also potentially toxic. Iron-overloaded fibrosis resulting from the accumulation of iron in the liver and the myocardium is a leading cause of morbidity and mortality in patients with iron overload. Nowadays, we have no effective prevention and treatment for iron-overloaded fibrosis.Deferoxamine(DFO), a traditional iron chelator, is a current mainstay of therapy for excessive iron deposition in patients. But, the cumbersome administration schedule and parenteral infusions of DFO lead to poor compliance of patients. Moreover, DFO is less effective for the treatment of iron-overloaded fibrosis. Recent studies have shown that calcium channels provide a major portal for iron uptake into cells, and calcium channel blockers(CCBs) can inhibit iron entry into cells and reduce the collagen volume in tissue。Salvia Miltiorrhiza(SM),a member of the Labiatae family, is highly valued for its dried roots or rhizomes in traditional Chinese medicine. As a well-known traditional medicine with promoting blood circulation effects, SM has shown versatile anti-fibrotic, anti-oxidative, anti-inflammatory and anti-apoptotic effects. Accumulating studies have demonstrated that the protective effects of SM on the cardiovascular system may be attributed partly to its functional property as a CCB by reducing intracellular Ca2+ concentrations. It is reported that SM also can reverse experimental hepatic fibrosis.Based on above information, we assumed that under iron overload condition, SM had protective effect on various organs. In the present study, we use techniques, such as pharmacology, molecular biology, and pathomorphology, to systematically study the following subjects:(1) Protective effects of Salvia Miltiorrhiza on iron-overloaded zebra fish,(2) Multitargeted inhibition of hepatic fibrosis in chronic iron-overloaded mice by Salvia Miltiorrhiza injection, and(3) Continuing treatment with Salvia miltiorrhiza injection attenuates myocardial fibrosis in chronic iron-overloaded mice. Part 1 Protective effects of Salvia miltiorrhiza injection on iron overload ebra fishObjective: To investigate the protective effect of Salvia Miltiorrhiza(SM) injection against the organ injury induced by iron overload in zebra fish.Methods: Acute iron overload model was established by given zebra fish ith ferrous sulfate in water. The lethal concentration of 50%(LC50) at ifferent times was calculated. Eighty fishes were randomly divided into ontrol, iron overload, 0.96 g/L SM, 1.28 g/L SM, 1.60 g/L SM and 1.92 g/L SM groups. The homogenates of fish liver and heart were collected to detect the changes of oxidative stress markers, glutathione(GSH), superoxide dismutase(SOD), and malondialdehyde(MDA).Results: LC50 of zebra fishes in 24 h iron overload group was 1.54 g/L, while LC50 in 48 h, 72 h and 96 h iron overload groups were 1.37 g/L. SM at 0.32 g/L, 0.64 g/L, 0.96 g/L, 1.28 g/L, 1.60 g/L and 1.92 g/L were added into iron resolion. The livability of zebra fishes were 0%,20%,50%,100%, 100% and 100% at 24 h,and were 0%, 0%, 30%, 50%, 90% and 100% at 96h. Treatment of iron-overloaded zebra fish with SM dose-dependently educed the fish mortality rate, and ameliorated the oxidative injury. SOD ctivity and GSH content of liver and heart in various SM treatment groups ere higher than that in iron overload group(P < 0.5). Meanwhile, MDA cntent of liver and heart in various SM treatment groups were lower than hat in iron overload group(P < 0.5).Conclusions: SM injection can significantly protect and treat the xidative injury induced by iron overload in zebra fish, and the effects of SM njection were in dose-dependent manner. Part 2 Multitargeted inhibition of hepatic fibrosis in chronic iron verload mice by Salvia miltiorrhizaObjective: To explore the effects and potential mechanism of Salvia Miltiorrhiza(SM) against hepatic fibrosis induced by chronic iron overload(CIO) in mice.Methods: Sixty male mice were randomized into five groups(n = 12 in each group): control(CONT, saline), chronic iron overload(CIO), low-dose SM(L-SM, 3 g/kg/day), high-dose SM(H-SM, 6 g/kg/day) and deferoxamine(DFO, 100 mg/kg/day) groups. The CIO model was established by intraperitoneal injection with iron dextran at 50 mg/kg body weight/day, and the entire course lasted for 7 weeks. The major constituents of SM injection were quantified by high performance liquid chromatography–ultra violet(HPLC-UV). Changes of hepatic iron, hydroxyproline(Hyp), GSH, SOD and MDA were assayed by standard procedures. Protein expression levels of type III collagen and tumor necrosis factor-α(TNF-α) were analyzed by immunohistochemistry, and m RNA levels of transforming growth factor-β(TGF-β), matrix metal proteinase-9(MMP-9) and caspase-3 were detected by RT-PCR. Morphological changes were observed with Prussian blue, Masson’s trichrome and hematoxylin-eosin staining.Results:(1) HPLC-UV showed that the actual concentrations of Danshensu, protocatechuic aldehyde and salvianolic acid B in the SM injection preparation were 2.15 mg/ml, 0.44 mg/ml and 1.01 mg/ml, respectively.(2) The liver coefficient was decreased by 28.30% in the L-SM group and 40.81% in the H-SM group as compared to the CIO group(P < 0.01).(3) Photomicrographs of liver sections by H&E staining showed that upon administration of L-SM and H-SM, the degree of pathological changes of the liver was markedly alleviated, as demonstrated by the restored lobular architecture, decreased hemosiderin deposition and fibrous tissue and limited inflammatory foci in the hepatic tissue.(4) The percentage area of Prussian blue staining was reduced by about 30.61% and 53.86% in liver sections with Prussian blue staining from the L-SM and H-SM groups, respectively, when compared to liver sections from the CIO group(P < 0.01).(5) After treatments of iron overload mice with L-SM and H-SM, fibrotic areas were decreased by about 46.50% and 89.29%, respectively, compared to the CIO group(P < 0.01).(6) The administration of SM at either the low dose or high dose evidently diminished the expression of type III collagen in the mouse liver.(7) After treatment of iron-overloaded mice with SM at a low dose and high dose, Hyp levels were observed at concentrations as low as 0.14 ± 0.01 μg/mg and 0.13 ± 0.01 μg/mg when compared with the CIO group(P < 0.05 and P < 0.01).(8) Compared with the CIO group, the TGF-β m RNA expression level was inhibited by both L-SM and H-SM by about 23.32% and 49.82%, respectively(P < 0.01); While, MMP-9 m RNA showed approximately 1.1-fold and 3.4-fold increases in the L-SM and H-SM groups, respectively(P < 0.01).(9) Compared with the CIO group, SM treatment in low-dose and high-dose elevated SOD activity and GSH content and reduced MDA level(P < 0.05 and P < 0.01).(10) TNF-α protein expression was weaker and less distinct in both L-SM and H-SM groups than that in the CIO group.(11) Compared to the CIO group, SM reduced the expression level of caspase-3 m RNA by about 18.25% at the low dose(P < 0.05) and 32.32% at the high dose(P < 0.01).Conclusions: SM displayed anti-fibrotic activity in the liver induced by chronic iron overload, which may be attributed to multitargeted inhibition of iron deposition and collagen accumulation, as well as oxidative stress, inflammation and apoptosis. Part 3 Inhibition of myocardial fibrosis in chronic iron-overloaded mice y Salvia miltiorrhiza injectionObjective: To investigate the effect and potential mechanism of SM on myocardial fibrosis induced by chronic iron overload(CIO) in mice.Methods: Kunming male mice(8 weeks old) were randomly assigned to six groups of 10 animals each: control(CONT), CIO, low-dose SM(L-SM), high-dose SM(H-SM), verapamil(VRP), and deferoxamine(DFO) groups. Only normal saline was injected in the CONT group. Meanwhile, mice in the other five groups were treated with iron dextran at 50 mg/kg per day intraperitoneally for 7 weeks, and those in the latter four groups also received corresponding daily treatments after 4 h iron given, including 3 g/kg or 6 g/kg of SM, 100 mg/kg of VRP, or 100 mg/kg of DFO. The iron deposition on heart tissue sections was estimated histologically using Prussian blue staining. Myocardial fibrosis was determined by Masson’s trichrome staining and Hyp quantitative assay. SOD activity, MDA content in heart homogenates were detedcted with colorimetry. The protein expressions of type I collagen(COL I) and type III collagen(COL III) were observed by immunohistochemistry. The expression levels of TGF-β1 and MMP-9 proteins were analyzed with Western blotting method.Results:(1) Heart coefficient declined by approximately 16.08% in the L-SM group and 18.47% in the H-SM group as compared to the CIO group(P < 0.01).(2) Photomicrographs of heart tissue sections with H&E staining showed that after treatment with L-SM or H-dose SM, levels of pathological changes were markedly alleviated, as demonstrated by the regularly arranged cardiomyocytes and decreased fibrous tissue.(3) Heart tissue sections with Prussian blue staining showed that SM treatment reduced the percent area of positively-stained tissue by approximately 38.64%(P < 0.05) at the low dose and 65.91%(P < 0.01) at the high dose, respectively, when compared with the CIO group.(4) Upon administration with SM, the positively-stained percent areas of fibrotic heart tissue were dose-dependently diminished by approximately 30.08% in the L-SM group and 64.37% in the H-SM group, as compared with the CIO group(P < 0.01).(5) After treatment with SM, the Hyp content decreased to as low as 0.89 ± 0.19 μg/mg in the L-SM group and 0.41 ± 0.20 μg/mg in the H-SM group(P < 0.01, vs. CIO group).(6) Treatment with SM elevated the SOD activity and lowered MDA concentration in a dose-dependent manner, when compared with the CIO group(P < 0.01).(7) SM treatment at either the low dose or high dose markedly reduced the positively-stained percent areas of COL I protein by approximately 48.35% or 63.87%, respectively, as compared to the CIO group(P < 0.01).(8) The expression level of TGF-β1 protein was reduced by SM treatment at both the low dose and high dose by approximately 31.33% and 73.02%, respectively, when compared with the CIO group(P < 0.01), while the expression level of MMP-9 protein was reduced by 25.11% and 71.99% respectively(P < 0.01).Conclusions: SM exerted activities against cardiac fibrosis induced by CIO, which may be attributed to its inhibition of iron deposition, as well as collagen metabolism and oxidative stress.