Role And Molecular Mechanisms Of Pulmonary Alveolar Proteinosis Induced By Indium Compounds Exposure

Objective To explore the biomarkers of indium-exposed workers,clarify the toxic effects of indium compounds in different forms,investigate the changes of pulmonary alveolar proteinosis（PAP）and biomarkers in rats induced by Nano-ITO,and verify the regulation of NF-κB/Nrf2 signal crosstalk on Nano-ITO-induced PAP in rats,the pathogenic mechanism of indium was preliminarily discussed in above four aspects to provide theoretical basis for the prevention of indium lung disease.Methods 1 A cross-sectional epidemiological study of indium-exposed workers from China was conducted to analyze the exposure level of airborne indium by individual and fixed-point sampling,evaluate associations between occupational indium exposure and serum biomarkers of early effect.Indium concentration in serum,urine,and airborne as exposure indices were measured by inductively coupled plasma-mass spectrometry.Serum biomarkers of pulmonary injury,inflammation,and oxidative stress were measured using ELISA.The associations between serum indium and serum biomarkers were analyzed to explore the mechanism of action of indium on pulmonary injury in indium-exposed workers.2 Animal experiments were conducted to measure inflammatory factors levels in bronchoalveolar lavage fluid（BALF）and lung tissue protein expressions in rats.Four different forms of indium compound-exposed rat models were established.Model I:0,1.2,3,and 6 mg/kg·bw ITO group;ModelⅡ:0,1.2,3,and 6 mg/kg·bw In₂O₃group;ModelⅢ:0,0.523,1.046,and 2.614 mg/kg·bw In₂（SO₄）₃group;ModelⅣ:0,0.065,0.65,and 1.3mg/kg·bw In Cl₃group.Lung pathological changes were assessed by hematoxylin&eosin（H&E）,periodic acid Schiff（PAS）,and Masson’s staining,and the protein changes were determined by immunohistochemistry.3 Dose-response（three divided doses）and time-course studies（six exposure durations）were performed to examine the pulmonary toxicity induced by Nano-ITO.At the end of the experiment,hematology and serum biochemical parameters were determined,and cytokines levels and oxidative stress were analyzed in the bronchoalveolar lavaged fluid.Rat lung tissues were also harvested for staining with H&E,PAS,Masson,and Sirius red.Ultrastructure of lung tissue cells was observed by transmission electron microscope.Nrf2 and NF-κB p65 in lung tissue were observed by immunofluorescence.The localization and expression of NF-κB p65,IκB-α,IKK-β,Nrf2,NQO1,HO-1 were observed by immunohistochemistry,Western blot and real-time fluorescent quantitative PCR.4 SFN+6 mg/kg·bw Nano-ITO and ATRA+6 mg/kg·bw Nano-ITO pre-treated intraperitoneally with SFN（5 mg/kg·bw）and ATRA（1 mg/kg·bw）twice per week before the administration of an intratracheal dose of Nano-ITO.Results 1 In the production workshop,the airborne indium concentration was 78.4±185.7μg/m³.The levels of serum indium and urine indium in indium-exposed workers were 39.3±25.6μg/L and 11.0±12.6 ng/g creatinine.Increased lung damage markers（LDH）,oxidative stress markers（T-AOC,CAT）,and inflammation markers（IL-1β,IL-6,TNF-α）were found in indium-exposed workers.Serum indium levels were statistically and positively associated with the serum levels of SP-A,IL-1β,IL-6 in indium-exposed workers.Among them,SP-A showed a time-response pattern.2 The results of animal experiments showed that,with an increase in dosage,indium exposure significantly increased the levels of serum indium and lung indium,as well as the BALF levels of IL?1β,IL?6,IL?10,and TNF?αand up-regulated the protein expression of SP-A,SP-D,KL-6,GM-CSF,NF-κB p65,and HO-1 in all rat models groups.No PAS-staining positive substance was found in the lung tissue of In₂（SO₄）₃and In Cl₃exposure groups,whereas ITO and In₂O₃rat models supported findings of pulmonary alveolar proteinosis and interstitial fibrosis seen in human indium lung disease.3 Nano-ITO intratracheal instillation caused pulmonary toxicity by inducing acute inflammation at 3 days,granuloma（nodule）formation and collagen hyperplasia at 14 days,and alveolar proteinosis at 56 days post-exposure.Pathological features of lung tissue include typical alveolar exudates,cellular fibrous nodules,enlarged alveolar fat droplet fusion,cholesterol crystal granuloma and pulmonary alveolar proteinosis.BALF analysis revealed significantly elevated levels of lung oxidative stress（SOD,TP,T-AOC,MDA,HO-1）,pulmonary injury（SP-A,SP-D,KL-6,GM-CSF,LDH）,and inflammatory markers（IL?1β,IL?6,IL?10,TNF?α）across most groups.Serum biochemistry results showed that Nano-ITO could affect the liver and renal functions of rats when exposed for 3 days.During the formation of pulmonary alveolar proteinosis induced by Nano-ITO,the levels of pro-inflammatory factors IL-1β,IL-6,IL-10,TNF-α,and Nrf2,NF-κB p65,IL-1β,TNF-αproteins expression were significantly up-regulated,and NF-κB p65,IKK-β.Nrf2 and its downstream proteins NQO1 and HO-1 were highly expressed in Nano-ITO-induced PAP rat,and showed a dose-dependent and time-dependent increase.4 SFN-treated reduced the protein and m RNA expression of NF-κB p65 and IKK-β,at the same time,it could further significantly promoted the up regulation of protein and m RNA expression of Nrf2,HO-1and NQO1（P<0.05）,and effectively inhibited the pulmonary interstitial fibrosis and protein deposition secondary to Nano-ITO-induced PAP in rats.On the contrary,ATRA inhibited Nrf2 and increased NF-κB p65 expression,activating NF-κB signal pathway further triggered oxidative stress and inflammatory reaction,which aggravated lung injury.Conclusions 1 Serum indium and urine indium can be used as specific exposure biomarkers to evaluate occupational indium exposure,serum markers SP-A,IL-1βand LDH can be used as sensitive indicators for screening early health damage of indium occupational exposure population.2 Exposure to four different forms of indium compounds can induce different degrees of inflammation in rat lung tissue,which were consistent with the results of the population;ITO and In₂O₃can induce pulmonary alveolar proteinosis.3 NF-κB/Nrf2 signal pathway is involved in the process of Nano-ITO-induced pulmonary alveolar proteinosis in rats.4 The current study demonstrated that SFN can effectively attenuate Nano-ITO-induced pulmonary alveolar proteinosis by activating Nrf2and inhibiting NF-κB signal pathway.Figure 72;Table 26;Reference 132...