| ObjectivePleurotus citrinopileatus has a variety of effects,can be used as medicine,can also be used as food.Polysaccharides,known as host immune enhancers,are also known as "biological response modulators"(BRMs)in the world.They have many biological activities,such as lowering blood glucose,inhibiting cancer cell growth,regulating human immunity,antiviral,antioxidant and antibacterial.The polysaccharide of Pleurotus citrinopileatus is one of the important nutritional components in Pleurotus citrinopileatus.At present,there are few studies on the polysaccharide of Pleurotus citrinopileatus.Only a few literatures have reported the effect of antibacterial,antioxidant and regulating immune function,but no comprehensive and in-depth studies on antibacterial and anti-inflammatory have been seen.Therefore,the in vitro bacteriostasis and anti-inflammatory effect of polysaccharides from Pleurotus citrinopileatus on mice infected with pneumonia by Staphylococcus aureus were studied systematically.In order to provide experimental basis for the research and development of polysaccharide antibacterial and anti-inflammatory health products of Pleurotus citrinopileatus.Methods1.To test the anti-bacterial effect of Pleurotus citrinopileatus polysaccharide in vitro,seven common pathogenic bacteria including Staphylococcus aureus,Escherichia coli,Bacillus subtilis,Bacillus cereus,Pseudomonas aeruginosa,Aspergillus niger and Aspergillus flavus were selected.In vitro,the diameter of the bacteriostatic ring was determined by Oxford cup method and the minimum inhibitory concentration(MIC)was determined to evaluate the anti-bacterial effect of Pleurotus citrinopileatus polysaccharide.2.To test the anti-bacterial effect of Pleurotus citrinopileatus polysaccharide in vivo,BALB/c mice were taken as the research object,and the suspension of Staphylococcus aureus was dropped into the nasal cavity to prepare the pneumonia mouse model induced by staphylococcus aureus.Then the mice were treated with the different concentrations of Pleurotus citrinopileatus polysaccharide(low dose:3.6mg/ml and high dose: 7.2mg/ml)through aerosol inhalation after intervention.The mouse serum,bronchoalveolar lavage fluid(BALF),trachea and lung tissue,and lung tissue protein,etc.were collected.The protection effect of Pleurotus citrinopileatus polysaccharide was detected by HE staining,ELISA kit,Western blot and inflammatory cell detection methods in pneumonia mice induced by staphylococcus aureus.Results1.In vitro the results of bacteriostasis:(1)The diameter of the bacteriostatic ring of Pleurotus citrinopileatus polysaccharide was detected by Oxford cup method.The results showed that compared with the positive control group,Pleurotus citrinopileatus polysaccharide had strong bacteriostatic effect on Pseudomonas aeruginosa,Staphylococcus aureus,Bacillus subtilis,Aspergillus flavus and Aspergillus niger,but had weak bacteriostatic effect on Escherichia coli and Bacillus cereus.Taking Staphylococcus aureus as an example,the diameter of the bacteriostatic ring produced by 0.5 mg/m L Pleurotus citrinopileatus polysaccharide treatment was 8.1 ± 1.2 mm,and the diameter of the bacteriostatic ring produced by the positive control streptomycin was 14.4 ± 1.6 mm;(2)The minimum inhibitory concentration of Pleurotus citrinopileatus polysaccharide against seven kinds of bacteria was detected by MIC method.The results showed that the MIC values of Escherichia coli,Pseudomonas aeruginosa,Staphylococcus aureus,Bacillus subtilis,Bacillus cereus,Aspergillus flavus and Aspergillus niger were 1 mg/m L,0.25 mg/m L,0.5 mg/m L,0.5mg/m L,1 mg/m L,0.125 mg/m L and 0.25 mg/m L,respectively.2.In vivo the results of bacteriostasis:(1)HE staining results showed that compared with the normal control group,obvious hyperemia,inflammatory exudates and bacterial colonies could be seen in the bronchial lumen of mice in the model mice.The trachea wall was significantly thickened,the normal structure of lung tissue disappeared.And obvious hyperemia,vasoinflammatory reaction,obvious thickening and disorder of alveolar wall,and a large number of inflammatory cells infiltrated could be seen in the pulmonary interstitium;However,the bronchial and lung tissue structure of mice in the high dose intervention group of Pleurotus citrinopileatus polysaccharide tended to be normal.Compared with the model group,the inflammatory lesions of bronchial and lung tissue were significantly improved,and there was no significant difference compared with the normal control group;But there was no significant change in the inflammatory lesions of the bronchus and lung tissue in the low-dose intervention group of Pleurotus citrinopileatus polysaccharide,and there was no significant difference between the low-dose intervention group and the model group;(2)The results of ELISA kits showed that: Compared with the normal control group,the cytokines IL-1β,IL-6,TNF-αand IL-17 contents in the serum and BALF supernatant of the model group mice were significantly increased.There was statistical difference(P<0.05)between normal control group and model group.However,the contents of these four inflammatory factors in the high dose intervention group of Pleurotus citrinopileatus polysaccharide were significantly lower than that in the model group,and there was no statistical difference compared with the normal control group and the positive control group;While IL-1β,IL-6,TNF-αand IL-17 contents in low-dose intervention group decreased slightly,but there was no statistical difference between the model group and the positive control group;(3)The results of inflammatory cells detection in BALF sediment by automatic blood analyzer: Compared with the normal control group,the number of total cells,lymphocytes and neutrophils in the model group were significantly higher(P<0.05);Compared with the model group,the number of various inflammatory cells in the high dose intervention group was significantly lower(P<0.05),and there was no significant difference compared with the normal control group and the positive control group;Compared with the model group,the number of various inflammatory cells in the low dose intervention group of Pleurotus citrinopileatus polysaccharide decreased slightly,but there was no statistical difference.Compared with the normal control group,the number of inflammatory cells increased significantly.And there was statistical difference between two groups(P<0.05);(4)The results of Immuno-histochemical staining showed that NLRP3 protein was mainly expressed in the epithelial cell layer of the trachea and lung interstitium in the model group mice;The results of Histochemical staining and Western blot quantitative analysis showed that compared with the normal control group,the expression of NLRP3 in the model group was significantly up-regulated,with a statistical difference(P<0.05);Compared with the model group,the expressions of NLRP3 in the positive control group and the high-dose intervention group of Pleurotus citrinopileatus polysaccharide were significantly down-regulated.But compared with the model group,the expression level of NLRP3 in the low-dose intervention group of Pleurotus citrinopileatus polysaccharide had no significant change.And there was no statistical difference.Conclusion1.Pleurotus citrinopileatus polysaccharide has good antibacterial effect on seven common pathogenic bacteria,especially on two kinds of fungi Aspergillus flavus and Aspergillus niger,and three kinds of bacteria Pseudomonas aeruginosa,Staphylococcus aureus and Bacillus subtilis;2.Pleurotus citrinopileatus polysaccharide can significantly alleviate the inflammatory reaction in infected pneumonia mice induced by staphylococcus aureus;... |
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