Anti-idiopathic Pulmonary Fibrosis Activity Of Crude Extracts Of Pheretima And Its Purified Protein

Background and object:Idiopathic pulmonary fibrosis(IPF)is a typical age-related irreversible fibrotic disease with high mortality.and the inflammatory response are closely related.At present,pheretima has a wide range of clinical applications for respiratory diseases,but its specific mechanism of action is still unclear.This subject systematically studied the anti-idiopathic pulmonary fibrosis activity and mechanism of the crude extract of fresh pheretima,further purified the crude extract of fresh pheretima protein,and preliminarily explored the anti-idiopathic pulmonary fibrosis activity of purified protein P2.Research methods:(1)Choose the crude extract of fresh pheretima was selected as the Phosphate buffered saline extract from fresh pheretima with liquid nitrogen.A mouse model of idiopathic pulmonary fibrosis induced by intratracheal instillation of bleomycin was established,and the effects of crude extracts of fresh pheretima fumigatus on lung injury and pulmonary fibrosis in mice with pulmonary fibrosis were detected by HE staining and Masson staining.;The expressions of α-SMA,Vimentin,Collagen I,Fibronectin and E-cadherin in serum before and after administration of the crude extracts of fresh pheretima chinensis were detected by RT-qPCR and Western blot.The expression of macrophages and neutrophils in the bronchoalveolar lavage fluid of mice with pulmonary fibrosis was analyzed by flow cytometry.The expression of inflammatory factor IL-2 was analyzed by Elisa kit.(2)Separating and purifying the crude extract of fresh pheretima by gel size exclusion chromatography to obtain fresh pheretima purified protein.Compositional and functional analysis of P2 protein by mass spectrometry and bioinformatics analysis.(3)The cytotoxicity of P2 protein on human embryonic lung fibroblasts and the proliferation of myofibroblasts were detected by CCK8-method,and the apoptosis of myofibroblasts was detected by flow cytometry.RT-PCR and Western blot were used to detect the mRNA and protein expressions of α-SMA,Vimentin and E-cadherin.Research results:(1)2.5mg/ml crude extract of fresh pheretima can inhibit the mRNA and protein expressions of α-SMA,Vimentin,Collagen Ⅰ and Fibronectin(P<0.05),and at the same time increase the mRNA and protein expressions of E-cadherin Expression(P<0.01),2.5mg/ml of fresh Guang Dilong crude extract can reduce the content of macrophages and neutrophils(P<0.01),and in a dose-dependent manner,1.25mg/ml of fresh The inhibitory effect ofcrude extract of fresh pheretima on IL-2 and other inflammatory factors was significant(P<0.05).(2)Five elution peaks were obtained by separating and purifying the crude extract of fresh pheretima by gel size exclusion chromatography,named P1(50-115 kDa),P1(50-115 kDa),P2(40-125 kDa),P3(15-100 kDa),P4(5-15 kDa)and P5(none),among which the second peak has the strongest UV absorption signal,and P2 was initially selected for subsequent experiments.Mass spectrometry analysis showed that under the detection conditions set in this experiment,the purified protein(P2)of fresh pheretima contained a total of 91 human proteins.It plays an important role in the regulation of inflammation and fibrosis.The results of cell viability experiments showed that the purified protein(P2)of fresh pheretima could inhibit the proliferation of myofibroblasts(P<0.01),and could reduce the mRNA and protein expressions of α-SMA and Vimentin(P<0.05).The mRNA expression and protein expression of E-cadherin were increased(P<0.01).The results of apoptosis flow cytometry showed that the purified protein(P2)of fresh pheretima.Analysis conclusion:This study preliminarily shows that t the crude extract of fresh pheretima has a certain effect on the treatment of idiopathic pulmonary fibrosis,which may be mainly related to reducing fibrosis markers,increasing anti-inflammatory activity and inhibiting TGF-β1/Smad2/3 pathway.the crude extract of fresh pheretima can easily and reliably obtain the purified protein(P2)of fresh pheretima by gel exclusion chromatography,and it has a certain anti-pulmonary fibrosis effect in vitro.