Protection And Mechanism Of Salvianolic Acid B And Salvianolate On Experimental Pulmonary Fibrosis

Idiopathic pulmonary fibrosis(IPF)is the most common interstitial lung disease characterized by myofibroblast accumulation,asmooth muscle actin(a-SMA)over-expression,excessive collagen deposition and extracellular matrix(ECM)remodeling.It is a devastating and progressive respiratory disease with a mean survival of 2-3 years from initial diagnosis due to irreversible loss of lung function.Unfortunately,there is still no effective therapy until now.Thus,there is an urgent need to look for effective drugs for IPF.Oxidative stress is one important molecular mechanism underlying fibrosis in a variety of organs,including the lungs.Recently,it's widely reported that reactive oxygen species(ROS)played an important role in kinds of pulmonary injuries.ROS mediate multiple cellular functions,including proliferation,apoptosis,migration and differentiation,all crucial events to tissue homeostasis and remodelling.Salvianolic acid B(SalB)is one of the major water-soluble compounds of Salvia miltiorrhiza(Danshen),which was one of the most popular Chinese herbs listed in the Chinese Pharmacopoeia and has been used for the treatment of ischemic cardiovascular and cerebrovascular diseases clinically in China for centuries.As the most bioactive component of salvianolic acid extracted from Danshen,SalB shows higher free radical scavenging activity than vitamin C,and its antioxidant capacity is in agreement with its protective effects against cell injury from oxidative stresses.SalB has been reported to protect against liver fibrosis in animals and patients,as well as to reduce experimental renal interstitial fibrosis in rats.In this study,the SalB was supposed to be strongly anti-pulmonary fibrotic which might also be due to its antioxidant capacity.The aim of the present study was to evaluate the effect of SalB on transfer growth factor?1(TGF-?1)-induced A549 and MRC-5 cell models and salvianolate on Bleomycin(BLM)-induced pulmonary fibrosis in Wistar rats,investigate the amelioration of oxidative/anti-oxidative balance induced by SalB and salvianolate in experimental pulmonary fibrosis models and identify underlying molecular mechanisms.The pulmonary fibrosis cell models were first established by TGF-?1-stimulation in A549 and MRC-5.The anti-fibrotic activity of SalB was evaluated through morphological analysis,cell immunofluorescence staining and western blotting.The resluts showed that SalB could significantly inhibit the changes of morphology and expression of fibrotic bio-markers in A549 and MRC-5 induced by TGF-?1.The intravenous BLM-injection in rats was performed to estabilish the animal fibrosis model.Hematoxylin-eosin,masson,immunochimical staining and western blotting were carried out to verify the anti-fibrotic activity of salvianolate in vivo.The results showed that salvianolate significantly ameliorated BLM-induced histological alterations,blocked collagen accumulations and reduced ?-SMA expression in lung tissues.Results of immunofluorescence staining and flow cytometry analysis showed that ROS production induced by TGF-?1 in A549 and MRC-5 was significantly inhibited by SalB.The protection of SalB and salvianolate against oxidative stress during fibrogenesis in experimental pulmonary fibrosis models was confirmed by glutathione and malondialdehyde detection.Results of immunofluorescence staining,western blotting and PCR indicated that SalB could up-regulate Nrf-2 in both protein and mRNA level and induce Nrf-2 nuclear translocation in MRC-5 cells,which might be the probable mechanism for the antioxidant capacity of SalB.Taken together,SalB and salvianolate presented remarkable anti-fibrotic activity in experimental pulmonary fibrosis models due to its antioxidant capacity.The probable mechanism was Nrf-2 up-regulation in in both protein and mRNA level.