| Background Candida albicans is a commensal organism that can be frequently encountered in the gastrointestinal tract,urogenital tract,mouth,skin and respiratory of healthy adults.Candida albicans is also an opportunistic fungal pathogen of humans and other mammals.It can switch from benign commensal organism to pathogenic pathogen in hosts under a variety of conditions,such as HIV/AIDS infection,excessive use of broad spectrum antibiotics,congenital immunodeficiency disease,chemotherapy,radiotherapy and immunosuppressive therapies.According to statistical analysis,invasive candidiasis was still mainly caused by Candida albicans infection from 1997 to 2016.About one-third of the patients with pulmonary candidiasis required admission to the intensive care unit or mechanical ventilation,and the overall in-hospital mortality were significantly higher as compared with patients with non-Candida albicans fungal infections.In addition,Candida colonization in the lower respiratory tract will increase the incidence of hospital-acquired pneumonia and the risk of drug-resistant bacterial infection.The prognosis of immunocompromised patients with Candida colonization is worse.Bronchopulmonary candidiasis is an important part of invasive candidiasis,but rarely proven by histology.Therefore,the mechanism of Candida albicans causing lung injury needs to be further studied in animal experiments.A number of studies found that Candida albicans infection increase the production of pro-inflammatory cytokines and chemokines.The inhibition of inflammatory signaling pathways significantly protects against inflammation-induced acute lung injury in rodent animals.NF-κB,MAPKs,PI3K/Akt and STAT3 pathways were important inflammatory signaling pathways.Nevertheless,it remains need to explore whether these inflammatory signaling pathways involve in Candida albicans infection induced acute lung injury in mice.Objective（1）To determine intraperitoneal injection of dexamethasone and cyclophosphamide can establish the immunosuppressive model.（2）To determine intratracheal instillation of Candida albicans can establish the infective model.（3）To investigate Candida albicans infection can induce acute lung injury in mice.（4）To investigate the mechanism of which Candida albicans infection induced acute lung injury in immunosuppressive mice.Methods（1）Candida albicans challenge:Candida albicans（ATCC SC5314）was grown in YPD medium overnight（30℃）,washed,suspended in endotoxin-free PBS then counted.Counts correlated directly with colony forming units（CFU）.Candida albicans suspensions was adjusted to about 106</sup>107CFU.（2）Establishment of immunosuppressive animal model:Immunosuppression was performed by intraperitoneal injection of dexamethasone（Dexa 10mg/kg）for ten days and cyclophosphamide（Cy 150mg/kg）on the first day.The normal group received intraperitoneal injection of the same dose of saline at the same time.（3）Establishment of infective animal model:The next day after the animal immunosuppression model was set up,CA and Dexa+Cy+CA group mice were intubated by intratracheal injection of 50μl of Candida albicans（106～107CFU/ml）after intraperitoneal injection of 10%chloral hydrate（0.3ml/100g）.In the Control and Dexa+Cy group,the mice were intubated by intratracheal injection of 50μl of normal saline（NS）after anaesthesia.（4）Flow cytometry analysis:The peripheral blood of Control and Dexa+Cy group was used to determine immunosuppressive state by flow cytometry analysis.（5）Histology:All mice were sacrificed 6h after Candida albicans infection and lung tissues were collected.The lower lobe of the right lung was cultured and the other part was immersed in formalin（10%）then dehydrated and embedded in paraffin.Sections（5μm）were stained with H&E.（6）The inflammatory cytokines in serum were detected by ELISA.IL-6,TGF-β,Kc and Mcp-1 m RNA in lung tissues were measured by RT-PCR.The protein expression of NF-κB、MAPKs、PI3K/Akt and STAT3signaling pathways were evaluated by Western-Blotting.The positive nuclear localization of NF-κB p65,NF-κB p50 and p-STAT3 were detected through immunohistochemistry.（7）Survival test:Animal death was observed until 7 d after Candida albicans infection.Results1.Immunosuppressive animal model establishment.After intraperitoneal injection of Dexa and Cy,the mice showed mental tiredness,malaise,inactivity,no appetite,dishevelled hair and weight loss.Flow cytometry showed that the proportion of lymphocytes,CD4+T and CD8+T cells and CD4+T/CD8+T ratio decreased.The results showed that immunosuppressive model was successfully constructed.2.Infection animal model established.PAS staining of paraffin section of lung tissue showed that Candida albicans spores could be seen in lung tissue,and the growth of Candida albicans could be seen in lung tissue culture.The animal model of Candida albicans infection was successfully constructed3.Candida albicans infection-induced acute lung injury in normal and immunosuppressive mice.The lung weight of CA group was slightly higher than that of Control group.The lung weight in Dexa+Cy+CA group were significantly higher than those in Dexa+Cy group.HE staining showed that the structure of alveoli was clear and intact and there were no inflammatory cells infiltration in Control and Dexa+Cy group.An infiltration of numerous inflammatory cells and thickening of the alveolar wall were observed in the lungs in Candida albicans-treated mice.Immunosuppression treatment significantly aggravated Candida albicans-induced infiltration of inflammatory cells and thickening of the alveolar wall in mice.4.Candida albicans infection up-regulated the expressions of pulmonary inflammatory cytokines and chemokines in normal and immunosuppressive mice.The m RNA levels of pulmonary IL-6,TGF-β,Kc and Mcp-1 were significantly increased after Candida albicans infection in normal and immunosuppressive mice.The levels of serum IL-6,IL-1βand IL-17 were significantly increased after Candida albicans infection and the levels of serum IL-1βand IL-17 were further increased after Candida albicans infection in immunosuppressive mice as compared with normal mice.5.Candida albicans infection activated pulmonary NF-κB signaling in normal and immunosuppressive mice.No difference was observed on the level of pulmonary IκBαamong four groups.However,the level of pulmonary p-IκBαwas significantly increased after Candida albicans infection.The levels of pulmonary NF-κB p50 and p65 were significantly increased after Candida albicans in normal mice.And the levels of pulmonary NF-κB p50and p65 were further increased in immunosuppressive mice as compared with normal mice.Nuclear translocation of pulmonary NF-κB p50 and p65 were further analyzed using immunohistochemistry.Results showed that nuclear translocation of NF-κB p50and p65 was mainly observed in the pulmonary epithelial cells of normal and immunosuppressive mice.The number of NF-κB p50 and p65 positive nucleus was increased in immunosuppressive mice as compared with normal mice after Candida albicans.6.Candida albicans infection activated pulmonary MAPKs signaling in normal and immunosuppressive mice.The results showed that there was no difference in the expression of p38,ERK1/2 and JNK among the four groups.Candida albicans infection has no effect on the expression of p-ERK1/2 and p-JNK proteins,but can significantly induce lung p38 phosphorylation in normal and immunosuppressive mice.7.Candida albicans infection activated pulmonary PI3K/Akt signaling in normal and immunosuppressive miceCandida albicans infection had no effect on the expression of Akt protein in the lungs.However,Candida albicans infection significantly induced pulmonary Akt phosphorylation in normal and immunosuppressive mice.The p-Akt level in the immunosuppressive group was slightly lower than that in the normal group after Candida albicans infection.8.Candida albicans infection activated pulmonary STAT3 signaling in normal and immunosuppressive mice.The results showed that Candida albicans infection significantly induced lung STAT3phosphorylation.Compared with normal mice,the lung p-STAT3 level of immunosuppressive mice was further increased after infection.In addition,the levels of nuclear ROR-γt and ROR-αin the lung were significantly elevated 6 hours after Candida albicans infection in normal and immunosuppressive mice.Immunohistochemistry showed that p-STAT3 positive nuclei were mainly observed in the pulmonary epithelial cells of normal and immunosuppressive mice.The number of p-STAT3 positive nucleus was increased in immunosuppressive mice as compared with normal mice after Candida albicans.Conclusion（1）Immunosuppressive animal model was established through intraperitoneal injection of dexamethasone and cyclophosphamide.（2）Infection animal model was established through intratracheal injection of Candida albicans.（3）Candida albicans infection up-regulated the expressions of pulmonary inflammatory cytokines and chemokines in normal and immunosuppressive mice.（4）Candida albicans infection induce acute lung injury may be through activating pulmonary NF-κB,MAPKs,PI3K/Akt and STAT3signaling pathways in mice... |
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