Construction Of Hvcn1 Gene Knockout Mice Using CRISPR/Cas9 System And Its Effect In Lung Injury Induced By Black Carbon Exposure

Objective:Black carbon（BC）is the key component of atmospheric fine particulate matter(PM_2.5).The adsorption of heavy metals will lead to various toxic effects and health risks due to the change of its physical and chemical properties.At present,the research on the health effects and toxicology of black carbon is an urgent problem to be solved in the research on the health effects of PM_2.5.The main function of voltage-gated proton channel Hv1（encoded by Hvcn1 gene）is to expel protons produced by NADPH oxidase（NOX）from the cytoplasm to maintain respiratory burst.Studies have shown that Hv1 is selectively and functionally expressed in lung tissue,but its role in lung injury induced by black carbon exposure has not been reported.In this study,Hvcn1 gene knockout（KO）mice were obtained by CRISPR/Cas9 and microinjection techniques,and wild-type（WT）and KO mice were exposed by carboxylated black carbon（c-BC）and c-BC-lead complexs（c-BC-Pb）to study the role of Hvcn1 gene in pulmonary inflammation in mice.Methods:KO mice were obtained by CRISPR/Cas9 technique and microinjection technique.The gene level was detected by sequencing and ordinary PCR;the m RNA level was detected by RT-PCR;the protein level was detected by immunohistochemical staining with Hv1 specific antibody.Eight-week-old WT and KO C57BL/6N male mice were exposed by nasal drip.The experiment was divided into six groups:Control WT,Control KO,c-BC WT,c-BC KO,c-BC-Pb WT and c-BC-Pb KO.The contents of albumin（ALB）,alkaline phosphatase（ALP）and lactate dehydrogenase（LDH）in bronchoalveolar lavage fluid（BALF）were detected by automatic biochemical analyzer,and the contents of inflammatory cytokines in BALF were detected by interleukin-6（IL-6）,tumor necrosis factor-α（TNF-α）,interleukin-8（IL-8）and C-reactive protein（CRP）kits.The contents of malondialdehyde（MDA）,superoxide dismutase（SOD）and transforming growth factor-β1（TGF-β1）were detected by ELISA kit,the pathological changes of lung tissue were detected by HE staining,The EMT markers（E-Cadherin and Vimentin）were detected by immunohistochemical staining,and the mean Integrated Optical Density（mean IOD）was calculated by Image Pro Plus software to semi quantify the expression of the two proteins in the lung.Results:The knockout efficiency of CRISPR/Cas9 technique based on the mixed injection of four sg RNAs and Cas9 protein into the cytoplasm of C57BL/6N mouse fertilized eggs achieved 100%.Finally,two kinds of KO mice（Mut1 and Mut2）were successfully constructed.Immunohistochemical staining showed that there was no positive signal of Hv1 protein in the lung tissue of KO mice.The analysis of mouse reproductive ability showed that the number of male mice in the inbred offspring of two kinds of KO mice was significantly more than that of female mice.There was no difference in body weight between WT and KO mice exposed to two kinds of BC.The contents of ALB and ALP in BALF of KO mice were significantly higher than those of WT mice,and the content of LDH was significantly higher than that of WT mice only after c-BC-Pb exposure.The contents of TNF-α,IL-6,IL-8 and CRP in BALF of KO mice were significantly higher than those of WT mice after c-BC-Pb exposure.The content of SOD in BALF of KO mice was significantly lower than that of WT mice,and the content of MDA was significantly higher than that of WT mice only after c-BC exposure.The content of TGF-β1 in BALF of KO mice was significantly higher than that of WT mice.The results of HE staining showed that the local alveolar wall of KO mice significantly thickened and inflammatory cell infiltration after c-BC exposure;the pulmonary alveoli of KO mice contained fibrin deposits and a large number of inflammatory cells after c-BC-Pb exposure.The results of semi quantitative analysis of immunohistochemical staining showed that compared with c-BC WT,the value of E-Cadherin mean IOD in c-BC KO group decreased significantly,and the value of Vimentin mean IOD increased significantly;compared with c-BC-Pb WT,the value of E-Cadherin mean IOD in c-BC-Pb KO group decreased significantly,and the value of Vimentin mean IOD increased significantly.Conclusion:Two kinds of Hvcn1 gene knockout mice were successfully constructed and the knockout efficiency was 100%in this experiment.The results of animal experiments showed that Hvcn1 gene knockout led to the increase of the contents of four inflammatory cytokines and three biochemical indexes in BALF,as well as oxidative damage,pathological changes and EMT of lung tissue,that is,Hvcn1 gene knockout aggravated lung injury of mice caused by two kinds of BC exposure.