Vitamin D Deficiency Exacerbates Bleomycin-induced Pulmonary Fibrosis Partially Through Aggravating TGF-?/Smad2/3-mediated Epithelialmesenchymal Transition

Object: The purpose of this study was to further explore whether vitamin D deficiency(VDD)aggravates bleomycin induced pulmonary fibrosis by animal experimentsMethods: The present study consists of two independent experiments.Experiment 1,we used male C57BL/6J mice(4-week old).All animals were kept with temperatures of 20–25°C and humidity of 50%.All animals were fed standard diet and water freely.The circadian rhythm of 12 h light and 12 h darkness was maintained.After one week of adaptation,40 mice were randomly divided into two groups: vitamin D deficient(VDD)and control(Ctrl).Mice of the VDD group were fed with vitamin D deficient fodder.Mice of the Ctrl group were fed with standard fodder.Five weeks later,mice of the Ctrl group and the VDD group were randomly divided into two subgroups,respectively(10 mice each subgroup): Ctrl,bleomycin(BLM),VDD,VDD+ BLM.In the BLM and VDD+BLM groups,animals were intratracheally instilled with BLM(1.5mg/kg).In the Ctrl and VDD groups,animals were treated with equal volume of saline in the same way.Experiment 2,wild-type(Cyp27b1+/+)and Cyp27b1 knockout(Cyp27b1-/-)mice were fed.Mating wild-type mice with Cyp27b1 knockout mice produced Cyp27b1+/+,Cyp27b1+/-and Cyp27b1-/-mice.The genotype was confirmed by real time polymerase chain reaction(PCR)analysis.All animals were fed with standard diet and water freely.The circadian rhythm of 12 h light and 12 h darkness was maintained.Six weeks after birth,mice each strain were randomly divided into 2 subgroups: Ctrl and BLM.In the BLM subgroup,mice were intratracheally instilled with BLM(1.5mg/kg).In the Ctrl subgroup,mice were intratracheally instilled with equal volume of saline.All animals were sacrificed 2weeks after BLM instillation.Serum 25(OH)D level was measured.The middle and lower lobes of right lung were sheared for histopathology and immunohistochemistry.The remainder tissues were collected for Western blots and realtime real time polymerase chain reaction(RT-PCR).Results: The relative weight of lungs was elevated in BLM-treated mice.Col1?1 and Col1?2,two collagen protein genes,were upregulated,and collagen deposition,as determined by Sirius red staining,was observed in the lungs of BLM-treated mice.Ecadherin,an epithelial marker,was downregulated.By contrast,vimentin and ?-SMA,two EMT markers,were upregulated in the lungs of BLM-treated mice.Pulmonary TGF-?/Smad3 signaling was activated in BLM-induced lung fibrosis.Further analysis showed that feeding VDD diet,leading to VDD,aggravated elevation of BLM-induced relative lung weight.Moreover,feeding VDD diet aggravated BLM-induced TGF-?/Smad3 activation and subsequent EMT in the lungs.In addition,feeding VDD diet exacerbated BLM-induced pulmonary fibrosis.Additional experiment showed that Cyp27b1 gene knockout,leading to active vitamin D3 deficiency,exacerbated BLM-induced pulmonary fibrosis.Moreover,Cyp27b1 gene knockout aggravated pulmonary TGF-?/Smad2/3 activation and subsequent EMT in BLM-induced lung fibrosis.Conclusion: VDD probably exacerbates BLM-induced pulmonary fibrosis partially through aggravating TGF-?/ Smad2/3-mediated EMT in the lungs.