| Background:In recent years,various types of chemical disinfectants begin to enter people’s vision with the gradual improvement of health requirements,and play an important role in production and human life.Guanidine compounds have been widely used in health care,family life and food industry because of their strong alkalinity,high stability and good biological activity.At present,chlorhexidine,polyhexamethylene biguanidine,polyhexamethylene guanidine(PHMG)and their derivatives are widely used and studied in China.Although PHMG is a representative guanidine disinfectant,most of the toxicity studies on PHMG focus on acute oral toxicity test,skin and mucosal irritation test,etc.,and there is no study on the respiratory toxicity of PHMG in China,and lack of relevant theoretical data.Objective:1.The purpose of this study was to investigate relationship between time-dependent and dose-dependent response of inhaled polyhexamethylene guanidine(PHMG)aerosol in mice,and further provide toxicological data for the risk assessment of polyhexamethylene guanidine;2.To investigate the function of TGF-β/Smad signaling pathway in pulmonary fibrosis induced by inhalation of PHMG aerosol in mice.Methods:1.50 C57BL/6N 5-7 weeks old mice were randomly divided into 5 groups according to body weight,including the control group(pure water),the control time-dependent response group(pure water),the low dose group(1.03 mg/m3),the low dose time-dependent response group(1.03 mg/m3)and the high dose group(9.09 mg/m3),half male and half female in each group.The PHMG aerosol was blown into the inhalation chamber by aerosol generator.The low-dose group and the high-dose group were exposed to PHMG aerosol for 4 h per day for a continuous exposure of 21 days.During the exposure period,the concentration of PHMG aerosol in the inhalation chamber was measured.The control time extension effect group and the low-dose time extension effect group were exposed to PHMG aerosol for 4 h every day for 21 days,and then stopped to allow the mice to breathe normal air for 21 days.The weight change of each group was measured every other day.The mice in each group were weighed 24 h after the last exposure.The cells in the bronchoalveolar lavage fluids(BALFs)were collected and differential cell counts were conducted under the microscope.The liver,heart,spleen,lungs,kidneys were removed and weighted.For pathological changes of lungs,the slice was stained with hematoxylin and eosin(HE)using standard procedures.Masson trichrome staining was performed to observe the pulmonary fibrosis changes.2.The content of TGF-β1 in the supernatant of BALF was determined by the standard ELISA protocols.After thawing mouse lung tissue,liquid nitrogen was used to grind it into powder,the total mRNA of mice lung tissue was extract by using Trizol reagent,Real-Time PCR was used to test the expression levels of fibrosis related factors COL1A1 and FN in the pulmonary.The expression levels of α-SMA and TGF-β1 in the lung tissues of mice was detected by Immunohistochemical(IHC)and immunofluorescence(IF),respectively.Western blot was used to detect the expression levels of Fibronectin,Smad2,Smad3,p-Smad2,p-Smad3,Smad4 and TGF-β1 in lung tissues of mice.Results:1.After inhaling PHMG for 3 weeks,the body weight and lung pathology of mice showed obvious dose response,that is,with the increase of exposure dose,various adverse reactions of mice were gradually aggravated:The concentrations of PHMG aerosol in the low dose group and high dose groups were 1.03 mg/m3 and 9.09 mg/m3,respectively.During the exposure period,the body weight of mice in the high-dose group decreased significantly,while that of mice in the low-dose group still increased,but the growth trend slowed down,and the last day body weight was lower than that of the control group.Compared to the low-dose group and the control group,the lung coefficient of the high-dose group was significantly increased(P<0.01),while the kidney coefficient and the heart coefficient were significantly reduced(P<0.05).Compared to the control and low-dose group,the number of total cells,macrophages,lymphocytes,neutrophils and eosinophils in BALFs of the high-dose group were significantly increased(P<0.05).However,the total number of cells was significantly reduced in the low dose group compared with the control group(P<0.01).Histopathology displayed that destruction of cilia in lung tissue,alveolar wall structure disorder,thickened pulmonary septal,visible congestion,and airway epithelial cells shedding in each exposure group,while these pathological changes were more serious in the high-dose group,even parenchymal changes were also observed.The pathological score of lung injury in the low and high dose groups were 4.47±0.33 and 4.83±0.16,which were significantly higher than that in the control group(1.58±0.09).A large number of collagen deposited in pulmonary interstitial and the periphery of bronchiole in each dose group was observed according to Masson trichrome staining,and collagen deposition was more obvious in the high-dose group.The expression level of α-SMA protein of lung tissue in mice in each exposure group was increased significantly detected by immunohistochemical staining.The results of enzyme-linked immunosorbent assay showed that the content of TGF-β1 in the supernatant of bronchoalveolar lavage fluid in the low and high dose groups were significantly increased,and the immunofluorescence staining results also showed that the TGF-β1 positive expression region was significantly increased as well.The expression levels of COLA1 and FN mRNA in lung tissues of mice in the high-dose group increased significantly than those in the control group(P<0.05).Compared to control group,the background Smad2 and Smad3 levels in expose group were decreased,and expression of Smad4,p-Smad3,p-Smad3/Smad3,p-Smad2,p-Smad2/Smad2,TGF-β1 and fibronectin increased significantly.These results illustrated that TGF-β/Smad signaling pathway is activated in the process of pulmonary fibrosis induced by PHMG and reaction has a dose response relationship.2.After inhaling PHMG continuously for 3 weeks and then giving normal air for 3 weeks,the body weight and pathological changes of the mice showed obvious time response,that is,after the cessation of inhalation of PHMG,the adverse effects continued to develop and worsen:The concentrations of PHMG aerosol in the low dose time-dependent response group was 1.03 mg/m3.During the period after exposure stopped,the body weight of mice in the prolonged effect of low dose group still showed an increasing trend,but the increasing trend slowed down,and the last body weight was lower than that in the control time-dependent response group,but higher than that in the low dose group.Compared with the low-dose group,the lung coefficient of mice in the low-dose time-dependent response group was significantly increased(P<0.01),and the heart coefficient was significantly lower than that in the low-dose and the control time-dependent response group(P<0.01).The total number of cells in the BALFs of the low-dose time-dependent response group was significantly lower than that of the control group,but was significantly higher than that of the low-dose group.Histopathology displayed that destruction of cilia in lung tissue,alveolar wall structure disorder,thickened pulmonary septal,visible congestion,and airway epithelial cells shedding in each exposure group,while these pathological changes were more serious in the low-dose time-dependent response group,even parenchymal changes were also observed.The pathological score of lung tissue injury in the low-dose time-dependent response group was 5.08±0.12,which was significantly higher than 4.47±0.33 in the low-dose group and 1.57±0.08 in the control time-dependent response group.A wide range of collagen deposition was observed in the lung tissues of the mice in the low-dose time-dependent response group,with obvious parenchymal changes observed around.Immunohistochemical results showed that the expression level of α-SMA protein in the lung tissue of low-dose time-dependent response group was significantly higher than that of the low-dose and the control time-dependent response group.Enzyme-linked immunosorbent assay and immunofluorescence staining showed significantly increased levels of TGF-β1.Compared with the control group,the expression levels of COL1A1 and FN mRNA in lung tissues of mice in the low-dose time-dependent response group were significantly increased after inhaling the PHMG aerosol for 21 days and inhaling normal air for another 21 days.The concentrations of p-Smad2,p-Smad3,Smad4,p-Smad2/Smad2 and fibronectin in the low-dose prolonged effect group were significantly higher than those in the low-dose prolonged effect group,indicating that the TGF-β/Smad signaling pathway is still activated even after the exposure is stopped,which makes the pulmonary fibrosis induced by PHMG present a certain persistence and irreversibility.Conclusions:1.The 21 days repeated inhalation of PHMG aerosol primarily resulted in pulmonary fibrosis,and there was a noteworthy dose-response and time-response relationship,namely with the increase of the inhalation dose,the changes of pulmonary fibrosis become more obvious.Even after stopping the exposure,the adverse reactions will continue to develop,which indicates that pulmonary fibrosis induced by inhaling PHMG is persistent and irreversible.It also provides an ideal model and reliable analysis method for the further study on the toxicity of PHMG in pulmonary fibrosis.2.Long term inhalation of PHMG aerosol could induce pulmonary fibrosis in mice.TGF-β/Smad signaling mediated the extracellular matrix remodeling involved in the development of pulmonary fibrosis induced by PHMG.The related drug development for the TGF-β signaling pathway may be effective for the pulmonary fibrosis induced by PHMG inhalation exposure. |
PDF Full Text Request