Osthole Inhibits Isoprenaline-induced Myocardial Fibrosis In Mice And Its Mechanisms

Objective: To determine whether osthole could inhibit the myocardial fibrosis (MF)induced by isoprenaline (ISO) in mice and to investigate its potential mechanisms.Methods: In vivo, Kunming mice were randomly divided into5groups, namely, thecontrol group, the myocardial fibrotic model group, othole80and40mg/kg groups, andcaptopril25mg/kg group. Three days after administration, the mice in the medicine-treatedand model groups were given ISO5mg/kg by hypodermic injection. From the followingday the dose of ISO was reduced to2.5mg/kg, which lasted for30days. Then, the mice inthe medicine-treated groups were still given medicine for a week. Finally, heart weight wasthen weighed and the cardiac weight index (CWI) was calculated. Hydroxyproline (Hydro)level in myocardium was determined by a colorimetric method. Hematoxylin and eosin(H&E) and Masson's trichrome staining were used to estimate the extent of MF andcalculate the collagen volume fraction (CVF). RT-PCR was used to measure themyocardial mRNA expressions of peroxisome proliferator-activated receptor α/γ(PPARα/γ), transforming growth factor β1(TGF-β1) and matrix metalloproteinase-2/9(MMP-2/9). Immunohistochemistry was used to measure the myocardial proteinexpressions of nuclear factor κB-p65(NF-κB-p65), TGF-β1and MMP-9.In vitro, cultured mouse cardiac fibroblasts (CFs) were pretreated with osthole (2.5²0μg/ml) for2h and then stimulated with angiotensin II (Ang II,10^-6mol/L) for24h. ThemRNA expressions of PPARα/γ, TGF-β1and MMP-2/9in CFs were examined withRT-PCR method, the protein expressions of NF-κB-p65and TGF-β1were detected byWestern Blot method, respectively. In the next experiment, we further investigated theeffects of osthole on gene expressions of TGF-β1and MMP-2/9as well as proteinexpressions of NF-κB-p65and TGF-β1in CFs stimulated with Ang II after pretreatmentwith PPARα/γ antagonists. Results:In vivo, compared with the model group, the CWI and Hydro content in themyocardial tissues in the osthole-treated groups were decreased(P<0.05or P <0.01). Theresults from H&E and Masson's trichrome staining showed that the content of collagen inthe myocardial tissues and the CVF were decreased obviously(P<0.01). The RT-PCRresults showed that osthole could upregulate myocardial PPARα/γ(P<0.05or P<0.01)andMMP-2/9(P<0.01)mRNA expressions and downregulate TGF-β1mRNA expression(P<0.05or P<0.01). The immunohistochemistry results showed that osthole coulddecrease the myocardial NF-κB-p65and TGF-β1protein expressions and increase theMMP-9protein expression(P<0.01).In vitro, the results from RT-PCR and Western Blot indicated that osthole couldincrease PPARα/γ and MMP-2/9mRNA expressions as well as inhibit TGF-β1mRNAexpression(P<0.01)and the NF-κB-p65and TGF-β1protein expressions(P<0.01orP<0.05)in cultured CFs after stimulation with Angâ…¡. And osthole-upregulated myocardialMMP-2/9mRNA expressions and osthole–downregulated myocardial TGF-β1mRNA,NF-κB-p65and TGF-β1protein expressions were reversed(P<0.01or P<0.05) after theCFs were pretreated with PPARα/γ antagonists.Conclusion: Osthole can inhibit MF formation induced by ISO in mice, and itsmechanisms may be related to the reduction of TGF-β1expression via the activation ofPPARα/γ and subsequent inhibition of NF-κB, and the increment of MMP-2/9expressionsin myocardial tissues.