Research On The Anti-pulmonary Fibrosis Effects Of Salvianolic Acid B And Tanshinone ?A Based On THP-1 And MRC-5 Cell Models

Objective: Pulmonary fibrosis(PF)is an interstitial lung disease characterized by interstitial inflammation and pulmonary fibrosis.The pathological feature of PF is excessive deposition of extracellular matrix.Clinical manifestations include dyspnea and loss of normal lung function.The evolution of PF can be divided into oxidative stress,alveolitis and fibrosis stages.Salvia miltiorrhiza Bunge mainly contains water-soluble salvianolic acids and fat-soluble tanshinone compounds,which are represented by salvianolic acid B and tanshinone ?A,respectively.In recent years,the preparation of salvianolic acids and tanshinone has shown a certain effect in treatmenting PF at clinical.However,the current research on the anti-PF of Salvia miltiorrhiza Bunge often chooses one of salvianolic acids and tanshinone to represent Salvia miltiorrhiza Bunge,and only studies one process of evolutions of PF,ignoring the potential synergy of salvianolic acids and tanshinone and the complexity of the PF pathogenesis.Therefore,based on the mechanism of pulmonary fibrosis,this study selected THP-1,MRC-5 and their co-culture models to study the anti-pulmonary fibrosis effect and synergistic effect of salvianolic acid B and tanshinone ?A,the two main medicinal components of Salvia miltiorrhiza Bunge.It is significant for the clinical application and development of Salvia miltiorrhiza Bunge preparations and provides theoretical basis for the treatment of PF.Methods:(1)THP-1 inflammation model was established by PMA combined with lipopolysaccharide(LPS),using the number of adherent cells and the content of interleukin-1 beta(IL-1?)secretion from cells.In order to study the effect of salvianolic acid B and sodium tanshinone II A sulfonate on inflammation,enzyme-linked immunosorbent assay(ELISA)and real-time fluorescence quantitative polymerase chain reaction(q RT-PCR)were used to detect the m RNA level and protein level of tumor necrosis factor-alpha(TNF-?)and IL-1?.(2)The MRC-5 pulmonary fibrosis model was established by using transforming growth factor-beta1(TGF-?1)and taking cell proliferation as an index.In order to study the effect of salvianolic acid B and sodium tanshinone II A sulfonate on pulmonary fibrosis,the effect of salvianolic acid B and sodium tanshinone ?A sulfonate on the proliferation of MRC-5 cells induced by TGF-?1 was detected by CCK-8 method.The ?-SMA protein was detected by immunofluorescence.Western blotting(WB)was used to detecte the ?-SMA,fibronectin and type 1 collagen ?1(COL1?1)proteins and q RT-PCR was used to detecte the level of m RNA of ?-SMA,fibronectin and COL1?1.(3)The co-culture model of THP-1 macrophages and MRC-5 cells was preliminarily established by Transwell chamber and LPS.The level of m RNA of ?-SMA in MRC-5 cells was detected by q RT-PCR to reveal the mechanism and the synergistic effect of the two components of Salvia miltiorrhiza Bunge on the multi-pathway of PF.Results:(1)The effect of salvianolic acid B and sodium tanshinone ?A sulfonate on the THP-1 macrophage inflammatory model showed that 100 ng/m L PMA could induce THP-1 cells to change from suspension cells to adherent THP-1 macrophages after 48 hours.After 24 hours of rest,a THP-1 macrophage inflammation model with high expression of IL-1? could be established combined with 0.5 ?g/m L LPS for 24 hours.The results of ELISA and q RT-PCR showed that salvianolic acid B and sodium tanshinone ?A sulfonate could not only reduce the amount of IL-1? and TNF-? protein secreted by inflammatory model cells,but also reduce the inflammatory model IL-1? and TNF from m RNA levels(P < 0.05).And from the data of the combined group,it can be preliminarily speculated that the two types of ingredients have no synergistic effect on anti-inflammatory.(2)The effects of salvianolic acid B and sodium tanshinone ?A sulfonate on MRC-5 pulmonary fibrosis cell model showed that 10 ng/m L of TGF-?1 could accelerate the proliferation of MRC-5 after 48 hours(P < 0.05),and up-regulate the protein and gene levels of pulmonary fibrosis markers ?-SMA,FN and COL1?1,thus establish MRC-5 pulmonary fibrosis model.The results of immunofluorescence showed that ?-SMA protein was expressed in all groups and was distributed in both cytoplasm and nucleus.Western blot results showed that the combined group significantly reduced the relative expression of ?-SMA protein(P<0.05),but not in the single drug group;except for the salvianolic acid B group,the positive drug group,the sodium tanshinone ?A sulfonate group and the combined group significantly reduced the relative expression of COL1?1 protein(P<0.05);except for the single drug group,the positive drug and the combination group also significantly reduced the relative expression of FN protein(P<0.05).The results of q RT-PCR showed that the two main components of Salvia miltiorrhiza Bunge and SB431542 as a TGF-?1 inhibitor could significantly reduce the relative m RNA expression of ?-SMA,FN and COL1?1(P<0.05).From the data of the combined group,it can be preliminarily speculated that the two components have synergistic effects in anti-pulmonary fibrosis.(3)The effects of salvianolic acid B and tanshinone II A on co-cultured pulmonary fibrosis model showed that THP-1 macrophages were obtained by treating THP-1 cells with 100 ng/m L PMA for 48 hours,then THP-1 macrophages and MRC-5 cells were co-cultured in Transwell chamber for 24 hours,and then induced by 0.5 g/m L LPS for 48 hours.After that the morphology of MRC-5 cells could be changed,and MRC-5 cells were transdifferentiated.In this way,PF model can be successfully established.The results of q RT-PCR showed that the relative expression of ?-SMA in MRC-5 cells in dexamethasone group,salvianolic acid group B,sodium tanshinone ?A sulfonate group and the combination group was significantly decreased(P < 0.05).These results suggestted that salvianolic acid group B and sodium tanshinone II A sulfonate group have good inhibitory effects on pulmonary fibrosis,and inhibit inflammation can inhibit the process of pulmonary fibrosis to a certain extent Conclusions: Salvianolic acid B and sodium tanshinone ?A sulfonate have a good inhibitory effect on M1 classical activated macrophage inflammation model and TGF-?1-induced MRC-5 pulmonary fibrosis model.It has anti-inflammatory and antipulmonary fibrosis activity and can act on PF through multiple pathways.Salvianolic acid B and tanshinone ?A,two main components of Salvia miltiorrhiza Bunge,have no synergistic effect on anti-inflammatory,but they have synergistic effect in inhibiting fibrosis.The specific situation needs further study.