Baicalein Attenuates Monocrotaline-induced Pulmonary Arterial Hypertension By Inhibiting Endothelial-to-mesenchymal Transition

Background and Objective:Endothelial cells(EC)dysfunction plays a central role in the initiation and progression of pulmonary arterial hypertension(PAH).Endothelial-to-mesenchymal transition(EndoMT)was shown lead to endothelial dysfunction in PAH.The primary agents improving EC fuction such as endothelin receptor antagonists,phosphodiesterase inhibitors and prostacyclin analogs have shown certain clinical effects in patients with severe PAH,however,they fail to reduce the high mortality rates.Patients with PAH have a dark future.Baicalein is a bioactive flavonoid extracted from the roots of Scutellaria baicalensis Georgi.It has been recently reported to prevent endothelial dysfunction and inhibit epithelial to mesenchymal transition(EMT),a biological process which has many regulatory pathways in common with EndoMT.The aim of this study was to investigate whether baicalein improves the endothelial function and attenuates PAH by inhibiting EndoMT in a well-established monocrotaline(MCT)-induced rat model.Methods:1.Forty Sprague-Dawley Male rats were randomly assigned into four groups: control group,MCT-exposed group,MCT-exposed group plus 50 mg/kg baicalein(50 mg/kg baicalein group)and MCT-exposed group plus 100 mg/kg baicalein(100 mg/kg baicalein group).PAH was induced by a single subcutaneous injection of MCT.Rats in the control group received a single injection of saline.At the end of 2 weeks after MCT injection,the animals were treated with baicalein or vehicle for an additional 2 weeks.Right ventricle systolic pressure(RVSP),right ventricular hypertrophy index(RVHI)and right ventricular mass index(RVMI)was measured at the end of 4 weeks after MCT injection.2.Masson's trichrome and Sirius-red staining were used for morphological observation of cardiopulmonary interstitial fibrosis,and immunohistochemistry staining was used to further assess collagen?in these tissues.collagen?and ?-smooth muscle actin(?-SMA)were assessed by Western blot analysis.3.In vitro vascular reactivity studies of pulmonary artery.4.The protein expressions of ?-SMA,vascular endothelial cadherin(VE-cadherin),N-cadherin,Vimentin,snail,slug,bone morphogenetic protein receptor 2(BMPR2)and nuclear factor-?B(NF-?B)were assessed by Western blot analysis.Results:1.Baicalein attenuates MCT-induced PAH in rats.The MCT-treated rats showed a significant elevation of RVSP,RVHI and RVMI compared with the control group.The RVSP,RVHI and RVMI were significantly reduced in the baicalein(50 and 100 mg/kg)treatment groups compared with the MCT-treated group.2.Baicalein attenuates MCT-induced pulmonary fibrosis in rats.Masson's trichrome and Sirius-red staining showed a prominent lung fibrosis in MCT-treated rats,The MCT exposure induced a significant increase in the expression levels of collagen?.However,when treated with baicalein(50 and 100 mg/kg),the expression of collagen?was significantly decreased and the pulmonary fibrogenesis were inhibited.3.Baicalein decreases collagen content in right ventricles(RVs)of MCT-treated rats.Masson's trichrome and Sirius-red staining showed a prominent RV fibrosis in MCT-treated rats and the levels of collagen?and ?-SMA were increased significantly.Baicalein treatment(50 and 100 mg/kg)attenuated the increases of collagen?and ?-SMA levels in RV.4.Baicalein increases pulmonary artery relaxation to acetylcholine(ACh)in MCT-induced PAH.The onset of vascular reactivity with increasing concentrations of phenylephrine(PE)did not differ among the experimental groups.The maximal relaxation induced by ACh was greatly reduced in MCT-exposed rats,compared to control rats.Baicalein treatment(50 and 100 mg/kg)increases pulmonary artery relaxation to ACh in MCT-induced PAH.5.Baicalein prevents the MCT-induced EndoMT.The Western blot analysis showed that the expression of VE-cadherin was significantly decreased while the expression of mesenchymal markers,including N-cadherin and Vimentin,was increased in the MCT-exposed rats,compared with that in the control group.Baicalein treatment(50 and 100 mg/kg)attenuated the decreases of VE-cadherin and the increase of N-cadherin and Vimentin induced by MCT.These results indicated that baicalein treatment inhibited the MCT-induced EndoMT process.6.Baicalein regulates the expression of snail,slug,BMPR2 and NF-?B in pulmonary arteries induced by MCT.The Western blot analysis showed that the expression of snail and slug was significantly increased compared with the control group.The expression of BMPR2 was reduced and the NF-?B levels were significantly increased.Baicalein treatment(50 and 100 mg/kg)attenuated the increase of snail and slug,the decreases of BMPR2,and inhibited MCT-induced up-regulation of NF-?B.Conclusions:1.Baicalein attenuates MCT-induced PAH and decreased PAH-related cardiopulmonary fibrosis.2.Baicalein attenuates PAH by inhibiting EndoMT.3.Baicalein inhibits EndoMT partially via NF-?B-BMPR2 pathway.