Citrus Alkaline Extracts Mitigate Pulmonary Fibrosis In Mice Via Inhibiting Fibroblasts Senescence

Objective:Pulmonary fibrosis(PF)is a rare lung disease with limited treatment options.Our previous study showed that Citrus alkaline extracts(CAE)had antifibrotic effects in bleomycin induced PF mouse model.However,the compounds of CAE and its underlying mechanism in PF were unclear.Hence,this study was designed to identify the compounds in CAE,eludicate the effecti'veness of CAE,and further investigate the antifibrotic mechanism of CAE in PF mouse model.Methods:1.Identification of compounds in CAE.HPLC and UPLC-MS were used to isolate and detect the compounds in CAE.2.Confirmation of effectiveness of CAE in vivo.Firstly,72 C57BL/6J mice were divided into six groups,blank,model,CAE(32,64,96 mg/kg/d),and pirfenidone(300 mg/kg/d).All the groups were exposed to bleomycin except blank.After exposure to bleomycin,mice were orally administrated with CAE from day 1 to day 28.All the mice were sacrificed on day 28.The survival and body weight were recorded.Lung tissues were stained with H&E and Masson and fibrotic score was analyzed.Fibronectin and a-SMA were detected by Western Blot.TGF-? and TNF-? in mouse serum were detected by ELISA.Secondly,to confirm the safety of CAE,30 normal mice divided into five groups were given higher levels of CAE(96,192,384 and 768 mg/kg/d)and sacrificed after 28 days.Liver tissues and mice serum were collected for H&E staining and liver function detection.3.Exploration of the antifibrotic mechanism of CAE via inhibiting fibroblast senescence.Firstly,immunofluorescence staining of p16,p21 together with ?-SMA and ?-galactosidase staining(SA-?-gal)were performed to find the CAE on the fibroblast senescence in vivo.Secondly,primary murine lung fibroblasts(PMLFs)and MRC-5 cells were used to the following studies.p16 and p21 protein level in senescent cells were detected by Western Blot and SA-?-gal both in PMLFs and MRC-5 cells.Immunofluorescence staining of p16 and p21 were detected in PMLFs.Thirdly,senescence-associated secretory phenotype(SASP)including Collagen ?,?-SMA,CTGF,TNF-?,PDGF-a,PDGF-b and MCP-1 were detected in senescent PMLFs and CAE treated senescent PMLFs by Western Blot and RT-qPCR.Thirdly,the influence of CAE on fibroblast senescence was detected when p53 was inhibited by PTF-?.Western Blot was used to detect p16,p21,?-SMA,CDK4 and Cyclin D1 in both PMLFs and MRC-5 cells.SA-?-gal was used to detect the number of senescent cells in both PMLFs and MRC-5 cells.Immunofluorescence staining was used to detect the p16 and p21 in PMLFs.Fourthly,the COX-2 was detected by western blot after stimulated by CAE in both PMLFs and MRC-5.Then the COX-2 was knocked down in PMLFs or inhibited by NS-398 or indomethacin in MRC-5 cells to evaluate the effect of CAE on fibroblast senescence.SA-?-gal was used to identify the senescent cells and western blot was used to detect p16,p21,p53 and a-SMA.Finally,mice lung were infected with lentivirus conjugated COX-2RNAi to interfere the COX-2 expression in lung.Lung tissues were stained with H&E and Masson.Immunofluorescence staining was used to stain p16,p21 and p53 in lung.SA-P-gal staining was utilized to identify the senescenct cells.Results:1.Five compounds were identified in CAE,including narirutin,hesperidin,sinensetin,naringin and synephrine.2.CAE could prolong the survival rate of bleomycin induced mouse model and increase their body weight.CAE improved the pathology of lung tissue,inhibited the collagen deposition and significantly decreased the fibrotic score(p<0.05).CAE significantly inhibited the fibronectin and a-SMA expression in lung tissue(p<0.05)and decreased TGF-(3 and TNF-a in mice serum(p<0.05).CAE did not bring damage to liver tissue and increase alanine aminotransferase,aspartate aminotransferase and alkaline phosphatase in serum(p>0.05).3.CAE significantly downregulated the p16 and p21 concomitant with a-SMA in vivo(p<0.05).In addition,CAE decreased the senescent cells in lung tissue.CAE could significantly inhibited the p16 and p21 protein expression and decreased the senescent cells in both PMLFs and MRC-5 cells(p<0.05).Moreover,CAE decreased the SASP including collagen I,a-SMA,CTGF,TNF-a,PDGF-a,PDGF-b and MCP-1 in senescent cells,significantly in collagen 1,a-SMA,MCP-1,PDGF-b(p<0.05).CAE revealed an add-on effect on PTF-a in inhibiting fibroblast senescence in decreasing the senescent cells(p<0.05)and downregulating the p16 and p21(p<0.05)both in PMLFs and MRC-5 cells.Further,when COX-2 was knocked down in PMLFs or inhibited by NS-398 and indomethacin in MRC-5 cells,the antisenescence effcect of CAE on fibroblast was abrogated.CAE could not decrease the senescent cells(p>0.05)and the protein expressions of p16,p21 and p53(p>0.05).Finally,the COX-2 in mouse lung was interfered with lentivirus conjugated COX-2RNAi,the antifibrotic effect of CAE was diminished(p>0.05),as well as the antisenescence effect.Conclusions:1.The CAE mainly contained flavonoids and alkaloids,including narirutin,hesperidin,sinensetin,naringin and synephrine.2.CAE could effectively mitigate the bleomycin induced pulmonary fibrosis in mice and did not cause liver toxicity.3.CAE prevent fibroblast senescence to ameliorate pulmonary fibrosis via COX-2/p53 signaling pathway.