Anti-Pulmonary Fibrosis Effects And Mechanism Of Dehydrocostus Lactone

Pulmonary fibrosis?PF?is the disease characterized by severe destruction of alveolar structure,diffuse alveolar inflammation and pulmonary interstitial fibrosis caused by various reasons.With the continuous development of industrialization,the number of PF cases has increased year by year.However,due to the lack of effective treatment,PF patients have poor prognosis and high mortality,which seriously impact on human health.Existing drugs that act on a single target or a single intracellular signal transduction pathway can't prolong the survival time and have no beneficial effects on improving the living quality of patients with PF.Pirfenidone and nidanib are currently commonly used drugs for the treatment of PF in clinical practice.However,both of them act on a single target.Moreover because of their severe side effects,they are not suitable for long-term treatment of PF patients.Some Chinese herbal medicines and their active ingredients have been wildly studied due to their characteristics of multi-target,multi-pathway and mild side effects in the treatment of PF.Dehydrocostus lactone?DCL?is an anthraquinone compound isolated from the dried roots of Costustoot.It has been shown that DCL has anti-inflammatory,anti-oxidant and other pharmacological effects.We also found that DCL attenuated bleomycin-induced inflammatory damage in the lungs of mice.This paper intends to use the in vivo and in vitro experimental PF model to investigate the pharmacological effects and corresponding mechanisms of DCL.Objective:The PF model induced by BLM and the PF model of HELF cells induced by TGF-?₁ in vitro selecting model were used to study the anti-PF effect of DCL and its molecular mechanism,respectively,which provided experimental and theoretical basis for the clinical treatment of PF with DCL.Methods:1.Effects of DCL on PF model of mice120 healthy male mice from Kunming were divided into 6 groups randomly:Control group,BLM group?BLM?,DCL low?DCLL?,DCL medium?DCLM?,DCLH high?DCLH?dose group and pirfenidone?PFD?,20 mice in each group.0.9%sodium chloride injection was instilled into the trachea in control group.While in the other groups,solution of BLM 5 mg/kg was instilled into the trachea to establish a pulmonary fibrosis model.On the second day,DCL 5,10 or 20 mg·kg^-1·d^-1 and PFD50 mg·kg^-1·d^-1 were administered intragastrically once a day for 21 days in treatment groups respectively.Both Control and BLM groups were given equal volume of normal saline.On the 7th,14th and 21st day after BLM,the body weight and the mortality of the mice were recorded.Lung tissue samples were taken and frozen at-80?for use.The morphology,inflammation and collagen accumulation of lung tissue were observed by HE and Masson staining.The expression of Collagen-I,Collagen-III and?-SMA in mice lung tissue was detected by immunohistochemistry and Western Blot;the content of hydroxyproline?HYP?in the tissues was examined by ELISA kit2.Effects of DCL on TGF-?₁-induced PF model on HELF in vitroThe normal cultured HELF were seeded into a 6-well plate as a density of 1×10⁵cells/mL,2 mL per well.After 24 hours of culture,the medium was discarded.HELF were divided into 6 groups randomly,Control,TGF-?₁?Model?,DCLL,DCLM,DCLH and PFD groups.All groups were cultured with 2 mL serum-free DMEM.Except for Control group,10?g/L TGF-?₁ was added to all other groups,and the serum was discarded after another 24 hours of culture.?1?Control group and?2?Model group were added with 2 mL serum-free DMEM;?3?-?5?groups,namely the DCLL,DCLM,and DCLH groups,add 2 mL containing 5,10,or 20?M DCL serum-free DMEM;?6?PFD group was added to 2 mL containing of 10?M PFD serum-free DMEM.24 hours later,the morphology of the cells was observed under the microscope.The viability was detected by MTT assay;the whole and nuclear protein of HELF were collected,Collagen-III,Collagen-I and?-SMA cells were detected by Western Blot;The expression of?-SMA in HELF cells were detected by immunofluorescence assay.3.Effects of DCL on oxidative stress levelOn BLM-induced mice PF and TGF-?₁–induced HELF model,flow cytometry was used to detect the effects of DCL on reactive oxygen species?ROS?levels in HELF.The content of GSH in mouse lung tissue,MDA and SOD content in mice serum were detected by ELISA kit.4.Effects of DCL on TGF-?₁/Smad₂ and NOX₄/Nrf₂ signaling pathwayProtein expression of TGF-?₁,p-Smad₂,NOX₄ and Nrf₂ on BLM-induced mice PF and TGF-?₁–induced HELF model were detected by Western Blot.Nrf₂ in nuclear portion was detected as well.5.Validation of predictive potential therapeutic factorThe normal cultured HELF were seeded into a 6-well plate as a density of 1×10⁵cells/mL,2 mL per well.When the cell density reached 70%-80%,medium was replaced with fresh antibiotic-free and serum-free medium before transfection.To investigate the effects of Nrf₂ siRNA on viability,extent of fibrosis and intracellular signaling pathway,HELF was divided into 4 groups:Control group,TGF-?₁ group?Model group?,TGF-?₁+negative control group,and TGF-?₁+Nrf₂ siRNA group.After Nrf₂ gene silencing,to investigate the effects of DCL on viability,extent of fibrosis and intracellular signaling pathway,HELF were divided into 5 groups:DCL experimental group:Control group,TGF-?₁ group?Model group?,DCL+TGF-?₁group,TGF-?₁+DCL+negative control group,TGF-?₁+DCL+Nrf₂ siRNA group.In each transfection group,cell culture was replaced with fresh antibiotic-free and serum-free medium after 4-6 h.48 hours later,the green fluorescence was observed under a fluorescence microscope to determine the transfection efficiency.Except for the Control group,10?g/L of TGF-?₁ was added to each group for 24 h to form a pulmonary fibrosis model,followed by 10?M DCL to each dish.Expression of Collagen-I,Collagen-III,?-SMA and Nrf₂ in HELF were detected by Western Blot.Results:1.DCL attenuated BLM-induced pulmonary fibrosis in miceGeneral condition of BLM mice was worse,while it much better in DCL treatment mice.HE staining results showed that the lung tissue structure of the BLM group was severely damaged with large area.During DCL treatment,the lung tissue structure and cell morphology recovered to normal gradually,and the alveolar septal proliferation was not obvious.Szapiel score data showed that the degree of alveolar inflammation in the DCL groups were reduced significantly?P<0.05?.Masson staining showed that a large amount of collagen fibers appeared in the lung tissue of the BLM group,and the degree of fibrosis was significantly reduced in each DCL treated groups.Compared with the BLM group,the expression of Collagen-I,Collagen-III and?-SMA protein in the lung tissue of the mice,the content of HYP decreased significantly in DCL treated groups?P<0.01?.Compared with DCLL,HYP content in group of DCLH was significant decreased?P<0.05?.2.DCL attenuated TGF-?₁-induced HELF cells fibrosis.MTT results showed that DCL showed no cytotoxicity in the experimental dose.Western Blot showed that Collagen-I and Collagen-III were decreased in each group compared with the model group.The expression of?-SMA protein in HELF cells was significantly lower in the control group and the different dose drug group than the group of Model?P<0.05?.3.Effects of DCL on oxidative stress levelsGSH in lung tissue of mice,MDA content,SOD activity in serum and intracellular ROS levels were detected.The results showed that compared with the BLM group,the MDA content in the DCLL,DCLM and DCLH groups decreased?P<0.05?,the SOD activity increased?P<0.05?,ROS levels reduced?P<0.05?,significantly.GSH levels increased significantly in both DCLM and DCLH groups?P<0.05?.4.Effects of DCL on TGF-?₁/Smad₂ and NOX₄/Nrf₂ pathwaysThe results showed that compared with BLM group,the expression of TGF-?₁,P-Smad₂ and NOX₄ in lung tissue of DCL group decreased?P<0.05?,Nrf₂ increased?P<0.05?,significantly.Compared with the TGF-?₁ model group,the expression of TGF-?₁,p-Smad₂ and NOX₄ decreased,in each DCL groups in HELF PF model?P<0.05?.Expression of Nrf₂ showed an increased in nuclear portion,but decrease in cytosol fraction?p<0.05?.5.Validation of predictive potential therapeutic factorResults showed that compared with the control group,the viability of the model group was increased?P<0.05?and the expression of Collagen-I,Collagen-III and?-SMA were increased remarkably.Compared with the modal group,the viability of the TGF-?₁+Nrf₂ siRNA group was higher.Treatment with DCL,compared with DCL+TGF-?₁,the viability in TGF-?₁+DCL+Nrf₂ siRNA group was higher than DCL+TGF-?₁,the expression of Collagen-I,Collagen-III and?-SMA were significantly increased?P<0.05?.Conclusion:1.PF in mice induced by BLM was attenuated by DCL significantly.2.PF in HELF induced by TGF-?₁ was inhibited by DCL significantly.3.On BLM-induced PF mice in vivo model and TGF-?₁-induced HELF PF in vitro model,DCL down-regulated the levels of MDA,ROS and up-regulated the levels of GSH,SOD.4.Both On BLM-induced PF mice in vivo model and TGF-?₁-induced HELF PF in vitro model,DCL down-regulated the expression levels of TGF-?₁,p-Smad₂,NOX₄ and up-regulated the expression of Nrf₂.5.After silencing Nrf₂ by transfection with Nrf₂ siRNA,DCL couldn't reduce the expression of Collagen-I,Collagen-III and?-SMA.