| Purpose:Paraquat(PQ)is a heterocyclic compound of bipyridine,which belongs to a kind of efficient,rapid,non-selective,highly water-soluble herbicide.PQ has been widely used in agriculture due to its good weeding effect and low price.However,PQ is highly toxic to humans and animals,and its toxicity progresses rapidly,resulting in multiple organs injuries after exposure.Currently,the mortality rate from PQ poisoning remains high due to the lack of effective treatment.Although the use of PQ has been banned in China,cases of suicide by taking PQ or poisoning caused by accidental exposure have occurred frequently,especially in remote rural areas.PQ causes multiple organs injury,and the main target organ is the lungs which induces acute lung injury(ALI)in the early stage,irreversible pulmonary fibrosis in the late stage,and eventually leads to respiratory failure and even death.Therefore,the search for alternative drugs to relieve the PQ-induced ALI remains a profound importance.Previous studies have shown that Traditional Chinese Medicine and its prescriptions may play an important role in alleviating the ALI induced by PQ.Andrographolide,as a diterpenoid compound which mainly extracted from the roots and stems of Andrographis Paniculata,has series of anti-inflammatory and antioxidant activities and widely used in the treatment of upper respiratory tract infection,rheumatism,arthritis and other diseases.Combined with relevant literature review,it was found that the effect of Andrographolide on reducing the PQ-induced ALI was complicated and unclear.Therefore,this article aims to investigate the effects of Andro on the PQ-induced ALI and its potential molecular mechanism,which would provide the experimental evidence support for alternative drugs to relieve the PQ-induced ALI.Methods:1.Andrographolide mitigates inflammatory reaction in the PQ-induced ALI by activating the Nrf2/HO-1 pathway.(1)An PQ-induced ALI model was established by using the SPF C57BL/6J mice.HE and Masson staining were used to evaluate the degree of PQ-induced ALI injury and collagen fiber deposition,as well as the effect of Andro treatment.(2)The wet/dry(W/D)proportion of the lung tissue was determined to evaluate the degree of PQ-induced pulmonary edema and the effect of Andrographolide treatment.(3)The levels of inflammatory factors IL-6,TNF-α and IL-1β in lungs were determined by the related ELISA kits to evaluate the inflammatory response.(4)The expression level of the Nrf2 protein in lungs was detected by the immunohistochemistry(IHC)method.(5)The protein expressions of Nrf2,NQO-1 and HO-1,and the protein levels of p65 NF-κB and p-p65 NF-κB were detected by Western Blot.(6)Mouse type II alveolar epithelial cells(MLE-12)were used as the research object,and the CCK-8 was used to screen the optimal concentration of PQ and Andrographolide,and the PQ-induced semi-inhibitory concentration(IC50)was used as the optimal concentration to establish an cell injury model.Andrographolide concentration was significantly improved with PQ-induced cell activity decline as the intervention concentration for the subsequent experiments.(7)Lentivirus transfection was used to knock down Nrf2 gene to reduce Nrf2 gene expression in MLE-12 cells,and the expression of p65 NF-κB,p-p65 NF-κB,IκBα and p-IκBα was detected by Western Blot.The concentrations of IL-1β,IL-6 and TNF-α in MLE-12 cells were determined by the ELISA kits.2.Andrographolide alleviates oxidative stress in the PQ-induced ALI by activating the Nrf2/HO-1 pathway.(1)An PQ-induced ALI model was established using SPF C57BL/6J mice,and SOD,CAT and MDA levels in lung tissues were determined by corresponding kits.(2)The level of Nrf2 in lungs was detected by the immunohistochemistry(IHC)method.(3)The protein levels of Nrf2,NQO-1 and HO-1 were detected by Western Blot.(4)Nrf2 gene was knocked down by lentivirus transfection method to lower Nrf2 gene expression in MLE-12 cells.The levels of NQO-1 and HO-1 in cells were detected.SOD,CAT and MDA levels in MLE-12 cells were measured by the kits.The intracellular ROS levels were detected by the reactive oxygen species(ROS)kit,and observed by fluorescence microscope.3.Andrographolide inhibits apoptosis in PQ-induced ALI by activating the Nrf2/HO-1 pathway.(1)An PQ-induced ALI model was established in the SPF C57BL/6J mice,and apoptosis of lungs was detected by the TUNEL assay.(2)The level of Nrf2 protein in lungs was detected by immunohistochemistry(IHC).(3)Protein expressions of Nrf2,NQO-1,HO-1,Bcl-2,Bax,caspase 3 and cleaved caspase 3 were detected by Western Blot.(4)The protein levels of p-PI3 K and p-Akt in mouse lung were detected by Western Blot.(5)PI3K inhibitor LY294002 inhibited PI3K/Akt signaling pathway,and the levels of p-PI3 K,p-Akt,Bcl-2 and Bax in MLE-12 cells were detected to explore the effect of Andrographolide’s regulation of the PI3K/Akt pathway on apoptosis.(6)Lentivirus knockdown Nrf2 gene in MLE-12 cells,and apoptosis was detected by flow cytometry.The protein levels of Nrf2,HO-1,p-PI3 K,p-Akt,Bcl-2,Bax,caspase 3 and cleaved caspase 3 were measured by Western Blot.Results:1.Andrographolide decreases the levels of inflammatory cytokines in reducing the PQ-induced ALI in mice through the Nrf2/HO-1 pathway.(1)Results of HE and Masson staining in lungs showed that the alveolar structure in the Control group and the Andrographolide alone group was complete,with no obvious congestion or bleeding in the alveolar,no inflammatory cell infiltration,and no obvious collagen deposition.Compared with the control group,alveolar congestion,hemorrhage and edema were obvious observed in the PQ group,with a large number of inflammatory cells infiltrated in the alveolar,alveolar structure obviously destroyed,alveolar septum thickened,and collagen deposited in large quantities.After the treatment of Andrographolide,the damage of alveolar structure was markedly reduced,the infiltration of inflammatory cells was decreased,the degree of alveolar congestion was improved,and the deposition of collagen was significantly declined.The lung tissue injury score and collagen deposition rate showed that compared with the Control group,the lung tissue injury score and collagen deposition rate in the PQ group were the highest,while the lung tissue injury score and collagen deposition rate significantly decreased after andrographolide treatment.(2)The Wet/Dry ratio in lungs of mice was used to evaluate the severity of the PQ-induced pulmonary edema.The results showed that compared with the Control group,the W/D ratio was significantly increased in the PQ group.After the treatment of Andrographolide,the W/D ratio of lung tissues decreased significantly in a concentration-dependent manner.(3)Results in vitro and in vivo showed that the levels of inflammatory cytokines including IL-6,IL-1β and TNF-α were significantly increased in PQ group compared with Control group.After treatment with Andrographolide,the levels of inflammatory factors IL-6,IL-1β and TNF-α in vitro and in vivo were significantly decreased.(4)The expression levels of Nrf2,NQO-1,HO-1 and p-p65 NF-κB in lungs were obviously increased compared with the Control group and Andrographolide alone group.Compared with the PQ group,Andrographolide significantly increased the expression levels of the Nrf2,NQO-1 and HO-1 proteins in lungs,and significantly decreased the level of the p-p65 NF-κB protein in a concentration-dependent manner.(5)Lentivirus transfection was used to knock down Nrf2 gene in MLE-12 cells,and it was found that Andrographolide in reducing the anti-inflammatory protein including p-p65 NF-κB and p-IκBα effects,and the enhancement of IκBα was weakened,which resulting in increased levels of IL-6,IL-1β and TNF-α in the MLE-12 cells.2.Andrographolide reduces oxidative stress in alleviating the PQ-induced ALI by regulating the Nrf2/HO-1 pathway.(1)In vitro,compared with the Control group,SOD and CAT levels in lung were significantly decreased and MDA levels in lung tissue were marked increased in the PQ group.Compared with the PQ group,SOD and CAT levels in lung tissue were significantly increased,and MDA levels were decreased after treatment with Andrographolide,,which played an antioxidant role.(2)Immunohistochemistry(IHC)showed that the Nrf2 protein level in PQ group increased significantly compared with the Control group.Compared with the PQ group,Andrographolide treatment significantly increased the level of Nrf2 in lungs.(3)Western Blot results showed that PQ obvously increased the levels of Nrf2,NQO-1 and HO-1 in lungs compared with the Control group.Compared with the PQ group,the levels of Nrf2,NQO-1 and HO-1 in lungs were significantly increased after Andrographolide treatment in a concentration-dependent manner.(4)The flow cytometry and fluorescence detection results showed that PQ significantly raised the intracellular ROS level compared with the Control group.Compared with the PQ group,ROS production markedly decreased with Andrographolide pretreatment.After lentivirus knockdown of Nrf2 gene in MLE-12 cells,ROS significantly enhanced.Compared with the Control group,the levels of NQO-1,HO-1 and MDA proteins in the PQ group were visibly increased,while the antioxidant proteins including SOD and CAT were distinctly decreased.Compared with the PQ group,intracellular antioxidant proteins NQO-1,HO-1,CAT and SOD were significantly increased after Andrographolide pretreatment,while the MDA level was obviously declined.When the Nrf2 gene was knocked down,the antioxidant proteins including NQO-1,HO-1,MDA and SOD in the Andrographolide group were significantly descended,while MDA was significantly increased.It was speculated that the antioxidant effect of andrographolide was mainly related to the regulation of Nrf2/HO-1 pathway to improve the activities of SOD and CAT,and reduce the levels of intracellular ROS and MDA.3.Andrographolide reduces the PQ-induced ALI by inhibiting apoptosis through activating the Nrf2/HO-1 pathway(1)The TUNEL method in mice was used to evaluate the inhibitory effect of Andrographolide on PQ-induced apoptosis.The results showed that compared with the Control group,the number of apoptotic cells in PQ group was significantly increased.However,after the intervention of Andrographolide,apoptotic cells decreased significantly in concentration-dependent manner.(2)Immunohistochemical analysis(IHC)showed that the level of the Nrf2 protein was higher than that of the Control group in lung.Moreover,the expression of the Nrf2 in lung tissue could be significantly increased with Andrographolide treatment.(3)Results of Western Blot showed that PQ significantly increased the levels of Nrf2,NQO-1 and HO-1 compared with the Control group,while the expression levels of Nrf2,NQO-1 and HO-1 proteins were significantly increased in lung tissues in a concentration-dependent manner after Andrographolide intervention.(4)In mice,Western Blot detected the p-PI3 K and p-Akt protein levels in lung tissues.Results showed that compared with the Control group,PQ significantly increased the p-PI3 K and p-Akt protein levels.While compared with the PQ group,Andrographolide significantly decreased the p-PI3 K and p-Akt levels.The difference was significant and concentration-dependent.(5)The protein expressions of Bcl-2,Bax,caspase 3 and cleaved caspase 3 were detected by Western Blot.The results showed that the ratio of Bcl-2/Bax in lungs of mice decreased significantly,but the expression of pro-apoptotic proteins caspase 3and cleaved caspase 3 were significantly increased when compared with the Control group.Compared with PQ group,Andrographolide significantly increased the ratio of Bcl-2/Bax,and decreased the expression levels of the caspase 3 and cleaved caspase 3proteins.(6)In vitro,lentivirus transfection technique was used to knock down Nrf2 gene expression in MLE-12,and the apoptosis of cells was detected by flow cytometry.The results showed that compared with the Control group,the PQ group could significantly increase the occurrence of apoptotic events.Compared with PQ group,Andrographolide pretreatment significantly reduced the incidence of apoptotic events.When the Nrf2 gene was knocked down,the effect of Andrographolide on PQ-induced apoptosis was weakened.The expression of anti-apoptotic protein Bcl-2and pro-apoptotic proteins Bax,caspase 3 and cleaved caspase 3 were detected by Western Blot.The results showed that,compared with Control,the ratio of Bcl-2/Bax was significantly reduced in the PQ group.There were significant differences in pro-apoptotic proteins including caspase 3 and cleaved caspase 3.Compared with PQ group,Andrographolide pretreatment significantly increased intracellular Bcl-2/Bax ratio,reduced the caspase 3 and cleaved caspase 3 expression levels.After Nrf2 gene knockdown,the effect of Andrographolide on the PQ-induced apoptosis was significantly weakened,with a significant difference.(7)In vivo,Western Blot analysis have showed that compared with the Control group,PQ stimulated the increase of the levels of p-PI3 K,p-Akt and Bax in MLE-12 cells,while induced the decreased of protein level of Bcl-2 with significant differences.Compared with the PQ group,Andrographolide obviously reduced the protein levels of p-PI3 K,p-Akt and Bax,and increased the protein level of Bcl-2 with statistical significance.After treatment with PI3 K inhibitor LY294002,the expression levels of p-PI3 K,p-Akt and Bax were distinctly decreased,while the expression level of anti-apoptotic protein Bcl-2 was significantly increased.(8)Western Blot showed that the levels of Nrf2,HO-1,p-PI3 K and p-Akt were increased in the PQ group compared with the Control group.Compared with the PQ group,Andrographolide pretreatment significantly increased the levels of Nrf2 and HO-1 protein,and obviously decreased the levels of p-PI3 K and p-Akt.After the Nrf2 gene was knockdown,Andrographolide’s ability to promote Nrf2 and HO-1 protein levels was significantly reduced,while the expression of p-PI3 K and p-Akt was markedly increased.Conclusion:1.In animal models of PQ-induced ALI,Andrographolide significantly reduces the degree of PQ-induced pulmonary edema with its possible mechanism involved in reducing inflammatory response,decreasing oxidative stress injury and inhibiting apoptosis.2.In terms of inflammatory response,Andrographolide inhibits the NF-κB pathway to reduce the levels of IL-6,IL-1β,TNF-α in the PQ-induced inflammatory response.Andrographolide inhibits the activation of NF-κB pathway to reduce IL-6,IL-1β and TNF-α levels in alleviating the PQ-induced ALI involved in activating the Nrf2/HO-1 signaling pathway.3.In terms of oxidative stress,Andrographolide could activate the Nrf2/HO-1signaling pathway to improve the levels of SOD and CAT antioxidant enzymes,and decrease ROS and MDA synthesis,and finally alleviates the PQ-induced oxidative stress injury.4.In terms of apoptosis,Andrographolide decreases the levels of pro-apoptotic protein Bax and apoptotic executive protein levels named caspase 3 and cleaved caspase 3,and promotes the level of anti-apoptotic protein Bcl-2 to inhibit the PQ-induced apoptosis by the activation of Nrf2/HO-1 pathway.5.Andrographolide inhibits the occurrence of apoptotic events by inhibiting the activation of PI3K/Akt signaling pathway to increase the Bcl-2/Bax ratio.6.Andrographolide inhibits the PI3K/Akt pathway and the occurrence of PQ-induced apoptotic events through the activation of Nrf2/HO-1 signaling pathway.In summary,this paper elaborated the mechanism by which Andrographolide alleviated PQ-induced ALI from the aspects of inflammation,oxidative stress and apoptosis,and emphasized the mechanism of Nrf2/HO-1 signaling pathway in regulating inflammation,oxidative stress and apoptosis,providing possible experimental evidence support for Andrographolide’s treatment of PQ-induced pulmonary toxicity.There provides a theoretical basis for clinical treatment and prevention of the PQ-induced ALI. |
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