Inhibitory Effect And Mechanism Of Celastrol On Pulmonary Inflammation Induced By Multi-walled Carbon Nanotubes

Objectives:Multiwalled carbon nanotubes（MWCNTs）are high-strength nanofiber materials with excellent electrical and thermal conductivity,high strength,and great toughness;it has broad application in the fields of new energy vehicles,composite materials,electronics,medical treatment,bioengineering,environmental protection,and aviation.MWCNTs have similar fiber characteristics and exposure patterns to asbestos.Asbestos is a clear category1 carcinogen that can induce human lung cancer and malignant pleural mesothelioma after long-term exposure.The scientific community and general public have expressed significant concern over the potential carcinogenic effects of MWCNTs on individuals,particularly those working in the production environment.As a result,effective measures to prevent health hazards from exposure to MWCNTs have become an urgent and essential topic of scientific research.Our previous research,alongside other studies,have demonstrated that MWCNTs are capable of inducing lung cancer when inhaled through the respiratory tract.Chronic lung inflammation plays a critical role in the development and occurrence of lung cancer,and preventing inflammation progression can help inhibit cancer formation.Key research has highlighted oxidative damage,reactive oxygen species（ROS）production,and the activation of the nuclear factor-kappa B（NF-κB）pathway as central factors in MWCNT-induced inflammation.Targeting these factors for intervention could provide novel options and strategies in occupational protection for those exposed to MWCNTs.Celatrol is a pentacyclic triterpenoid that occurs naturally and is extracted from the roots of traditional Chinese medicine Tripterygium wilfordii.The compound exhibits multi-target biological effects,including antioxidation,anti-inflammation,and anti-angiogenesis.Notably,celatrol offers significant advantages in terms of bioavailability and sustained efficacy compared to other plant-based drugs.Furthermore,it has shown to be effective in preventing colitis-associated rectal cancer induced by sodium glucan sulfate in mice through antioxidant and NF-κB pathway inhibition.Given these promising results,it is worthwhile to investigate whether celastrol can also offer protective benefits against lung cancer caused by MWCNTs.The primary aim of this study was to examine the inhibitory impact of celastrol on oxidative stress and inflammatory harm triggered by MWCNTs,while also conducting a preliminary exploration into its potential mechanism.The outcomes of this research furnish a sound scientific and theoretical foundation for the effective implementation of preventative measures to counteract the cancer-causing effects of MWCNTs.Consequently,we hope that the research will help offer a fresh option for occupational protection to safeguard individuals exposed to MWCNTs.Materials and methods:In this study,the cell model of MWCNTs induced oxidative stress injury in BEAS-2B cells was established in vitro,followed by an animal model of MWCNTs induced chronic lung inflammation in C57BL/6 mice by bronchial perfusion.The inhibitory effect and molecular mechanism of celastrol on oxidative stress injury and chronic inflammation induced by MWCNTs were investigated which were used in vitro and in vivo models.All animal experiments were approved by Committee on Ethics of Biomedicine of Second Military Medical University（SMMU-20180432）.1.Test subjects:MWCNTs with outer diameter（20-30 nm）and length（10-30μm）were used and treated with BSA dispersion and ultrasonic depolymerization before exposure.2.Experimental subjects:BEAS-2B cells（bronchial epithelium transformed with Ad12-SV40 2B）,wild-type C57BL/6 mice and gene knockout(P50^-/-)C57BL/6 mice,male.3.Experimental dose and grouping:（1）In vitro experiment1)Dose:The toxicity dose of MWCNTs model group was 50μg/m L,and the intervention dose of celastrol was 0.25μM,0.5μM and 1μM.2)Experimental groups:normal control group,MWCNTs group,MWCNT+celastrol group,celastrol group.（2）Animal experiments1)Dose:The toxicity dose of MWCNTs was 4.5 mg/kg,and the intervention dose of celastrol was 2 mg/（kg·d）.2)Experimental groups:normal control group,MWCNTs group,MWCNTs+celastol group,MWCNTs+gene knockout(P50^-/-)group.4.Experimental methods and contentsThe main research contents are described as follows.In vitro experiment:（1）In the course of conducting in vitro cell tests,employing BEAS-2B cells,a study was undertaken to investigate the interplay between MWCNTs and celastrol over a period of 24 hours.The assessment of the cytotoxic effects and ROS production was carried out using CCK8 and DCFH-DA probes,respectively.The outcome of these investigations allowed for the identification of both the optimal dose of oxidative stress generated by MWCNTs and the requisite dosage of celastrol needed for intervention.（2）The PI reagent and DCFH-DA probe were enlisted to identify cellular mortality and ROS generation following a 24-hour intervention of celastrol.The objective was to assess the potential inhibitory effect of celastrol against the cytotoxicity provoked by MWCNTs.（3）Nuclear and plasma proteins were extracted and subjected to Western blot assay to evaluate the expression differences of KEAP1,NRF2,and HO-1 proteins among diverse groups.The aim of the study was to determine the inhibitory effect and mechanism of celastrol on the oxidative damage caused by MWCNTs.（4）The KEAP1 and NRF2 genes were transfected using lentivirus to induce silencing,and subsequent analysis involved the detection of cytotoxicity,ROS levels,and protein volume downstream of the silence genes using CCK8 and DCFH-DA probes,in addition to Western Blot analysis.The aim of this investigation was to determine the influence of the KEAP1/NRF2/HO-1 signaling pathway on oxidative stress induced by MWCNTs.（5）The RT-PCR technique was utilized to determine the expression level of the KEAP1 gene.Subsequently,protein synthesis inhibitors such as cycloheximide（CHX）and degradation inhibitors including MG132 and baffolomycin A1（Baf-A1）were employed to examine any alterations in the intracellular KEAP1 protein expression.Moreover,to elucidate the precise degradation pathway of KEAP1 protein triggered by celastrol,further investigation was conducted.（6）Through assessment of ROS production across distinct cellular cohorts subsequent to silencing the NRF2 gene,scrutinizing the NRF2 protein expression within the nucleus and total HO-1 protein expression within the entire cell,the pivotal target role of NRF2protein in the suppression of MWCNTs-induced oxidative damage by celastrol was examined.Animal experiments in vivo:（7）The present study delved into the inhibitory potential of celastrol with regards to the oxidative damage caused by MWCNTs.This was done by scrutinizing the levels of antioxidant enzymes GSH and SOD,as well as the expression of antioxidant protein HO-1,in lung tissues of wild-type mice.（8）The present study involved the construction of P50^-/-mice,followed by the utilization of a hematology analyzer to detect the contents of leukocytes,lymphocytes,and neutrophils in the alveolar lavage fluid from both wild-type and gene knockout mice.Additionally,the expressions of IL-1β,IL-6,and TNF-αwere assessed through ELISA,while lung tissue damage was evaluated through H&E staining.To determine the proportion of inflammatory cells present in the lung tissue,tissue fax scans were conducted.The study also examined the inhibitory effects of celastrol and P50 gene knockout on MWCNTs-induced lung inflammatory damage in mice.（9）In order to investigate the mechanism by which celastrol inhibits lung inflammatory injury induced by MWCNTs in mice,the expression of P50 protein,P65protein,and phosphorylated P65 protein was detected in lung tissues across all groups through the utilization of Western blot assay.This analysis was utilized to further examine the role of the NF-κB signaling pathway.Results:Through the above experiments,the following research results are obtained:1.The production of cellular ROS increased,and cell viability decreased upon treatment of BEAS-2B cells with MWCNTs,indicating that MWCNTs induce cytotoxicity through the overproduction of ROS.2.After the intervention of celastrol in the treated cells of MWCNTs,cell ROS level and cell death decreased.The results showed that celastrol could reduce the cytotoxicity induced by MWCNTs by inhibiting ROS level.3.After the intervention of celastrol in MWCNTs treated cells,KEAP1 protein expression in cytoplasm decreased,NRF2 protein increased,KEAP1 protein expression in nucleus decreased,NRF2 protein expression in nucleus increased,and total HO-1 protein expression in whole cell increased.The results showed that celastrol promoted NRF2protein into the nucleus by inhibiting KEAP1 protein expression and stimulated the expression of antioxidant protein HO-1 to play an antioxidant role.4.After KEAP1 gene silencing,ROS level decreased,cytotoxicity induced by MWCNTs was weakened,cytoplasmic NRF2 protein entry into the nucleus increased,and total HO-1 protein expression increased.After silencing NRF2 gene,ROS level was increased,cytotoxicity induced by MWCNTs was enhanced,and total HO-1 protein expression was decreased.The results show that KEAP1/NRF2/HO-1 signaling pathway regulates the oxidative damage induced by MWCNTs.5.After intervention with celastrol,KEAP1 m RNA expression increased,and total KEAP1 protein expression decreased.After inhibiting protein synthesis,KEAP1 protein degradation increased.After inhibiting ubiquitin-proteasome pathway,KEAP1 protein degradation was not inhibited.Inhibition of lysosome pathway inhibited KEAP1 protein degradation.The results showed that celastrol promoted KEAP1 protein degradation through lysosome pathway.6.After the silencing of NRF2 gene,the ROS production did not change significantly after the intervention of celastrol in the cells treated with MWCNTs.After intervention with celastrol,the expression of NRF2 protein and total HO-1 protein in the nucleus did not increase significantly.The results suggested that the silencing of NRF2 gene weakened the scavenging effect of celastrol on MWCNTs induced excessive ROS level in cells,and the key target of the antioxidant effect of celastrol might be NRF2 protein.7.A study was conducted to examine the inhibitory effect of wild-type mice on oxidative damage induced by MWCNTs.This involved the measurement of antioxidant enzymes GSH and SOD and the expression of antioxidant protein HO-1 in the lung tissue after a 4-week feeding period.8.After the treatment of MWCNTs,the contents of leukocytes,neutrophils and lymphocytes in the alveolar lavage fluid of mice were significantly increased,and the expressions of inflammatory cytokines IL-1β,IL-6 and TNF-αwere also significantly increased.The pathological injury of lung tissue was aggravated,and the number of leukocytes,neutrophils and lymphocytes were significantly decreased after the intervention of celastrol.The expression of inflammatory factor TNF-αalso decreased significantly,and the pathological injury of lung tissue was alleviated.After P50 gene knockout,the number of neutrophil granulocytes was significantly decreased,the expression of inflammatory factor IL-1βwas decreased,and the pathological injury of lung tissue was reduced.The proportion of inflammatory cells in lung tissue decreased.The results showed that both of the knockout of P50 gene and celastrol could inhibit the lung inflammation induced by MWCNTs in mice.9.After the mice were treated by MWCNTs,the expression of P50 protein and phosphorylated P65 protein was increased in the lung tissue of mice,and the expression of total P50 protein,P65 protein and phosphorylated P65 protein were decreased after celastrol intervention as well as P50 knockout.The results suggest that both of the celastrol and P50 gene knockout can prevent the activation of NF-κB signaling pathway induced by MWCNTs.Conclusions:1.Celastrol promotes the degradation of KEAP1 protein through the lysosome pathway and releases NRF2 protein,thus increasing the entry of NRF2 protein into the nucleus from the cytoplasm,binding with the antioxidant element in the nucleus,and stimulating the increase of the expression of downstream antioxidant proteins.2.The KEAP1/NRF2/HO-1 signaling pathway is effectively regulated by celastrol,resulting in the clearance of excess ROS caused by MWCNTs.Additionally,celastrol significantly inhibits oxidative damage caused by MWCNTs,thereby reducing their associated cytotoxicity.3.In mice,targeting the P50 gene through knockout measures has been found to effectively prevent the activation of the NF-κB signaling pathway,subsequently reducing the expression of inflammatory factors which in turn inhibits MWCNT-induced lung injury.4.Celastrol inhibits MWCNTs induced lung tissue inflammation by blocking the activation of NF-κB signaling pathway and reducing the expression of inflammatory factors.