| Acute lung injury(ALI)is clinically characterized by excessive uncontrolled inflammatory responses that can lead to acute respiratory distress syndrome(ARDS),which ultimately limits the therapeutic strategies.ALI has high morbidity and can cause high mortality both in humans and animals.Staphylococcus aureus(S.aureus)is a versatile and harmful pathogen,which is an opportunistic,Gram-positive bacterium.It is non-motile,non-spore forming bacteria and has ability to cause variety of diseases including ALI.Mouse model has been extensively used in ALI research for humans and other animals.We discovered that S.aureus is one of the most important pathogen,which cause serious lung injury with inflammation and cell apoptosis in cattle acute pneumonia tissues.Moreover,molecules belonging to inflammatory response,oxidative stress and apoptosis are abundantly expressed in these tissues.Ginsenoside Rb1(Rb1),an active ingredient obtained from Panax ginseng,possesses a broad range of medicinal and pharmacological properties,particularly anti-inflammatory,anti-oxidant,anti-apoptotic and anti-tumor activities.By virtue of these properties,huge research potential lies in this area.Therefore,the current research has been aimed to investigate pathogenesis of cattle pneumonia,and to explore protective effects of Rb1 on inflammation,oxidative damage,apoptosis as well as their molecular mechanism in ALI via mice model and murine macrophages RAW264.7 cells induced with S.aureus.1.The histopathological,etiological diagnosis and detection of relative molecules of some signaling pathways in cattle lung tissueRespiratory diseases are very much important in cattle,and is multi-factorial in etiology.S.aureus is a pathogen that is present as normal flora of skin and nares of cattle.The present study has been planned to explore the immunological mechanism of cattle lung in the natural colonization of S.aureus.Twenty lung samples of cattle were collected from abattoir located in Wuhan City,Hubei,China.Histopathologically,thickening of alveolar wall,accumulation of inflammatory cells in perivascular space,hyperemia,hemorrhages and edema were observed in infected lungs as compared to healthy lung samples.S.aureus was isolated from the infected lung tissue on differential selective media(Sheep blood agar,Mannitol salt agar and Staphylococcus Selective Agar No.110).Furthermore,molecular confirmation was carried by amplification of the S.aureus-specific nuc gene from the infected lung samples to identify S.aureus.Moreover,the relative m RNA and protein expression of NF-k B,mitochondria-mediated apoptosis pathway and TLR2 genes were significantly(p < 0.05)up-regulated in infected lung samples as compared to healthy lung samples.The Bcl-2 and Nrf2 pathway genes were downregulated significantly(p < 0.05)in lungs infected with S.aureus as compared to healthy lung samples.Conclusively,the present study has shown insights into the active immune response of host due to S.aureus in the lung tissue of cattle.2.Ginsenoside Rb1 Ameliorates S.aureus-induced Inflammation,Oxidative stress and Apoptosis in RAW264.7 CellsThe purpose of this study was to investigate the principle underlying mechanism against S.aureus-induced inflammation,oxidative stress and apoptosis as well as to evaluate the protective effects of Ginsenoside Rb1 in RAW264.7 cells.The RAW264.7 cells were divided into five groups,as the control group,S.aureus group,S.aureus+Rb1(10 μg/m L)group,S.aureus+Rb1(20 μg/m L)group and Rb1 group(20 μg/m L).The results of the cell viability assay revealed that there was no toxicity to RAW264.7 cells with different doses of Rb1 used in our experiment.The results of quantitative real time polymerase chain reaction(RT-q PCR)indicated that Rb1 significantly(p < 0.05)repressed interleukin(IL)-1beta(β),IL-6,tumor necrosis factor-alpha(TNF-α)in RAW264.7 cells stimulated by S.aureus.The immunofluorescence results indicated that Toll-like receptor 2(TLR2)was activated by S.aureus,and was significantly(p < 0.05)ameliorated by administration of Rb1.The expression of NF-k B and MAPK signaling pathways determined by western blot assay,indicated that Rb1 significantly(p < 0.05)attenuated the phosphorylation of NF-k B-P65,IKB-α,JNK as well as ERK proteins.The 20 μg/m L Rb1 dose group was used to explore protective effect of Rb1 on the oxidative stress and apoptosis.It was also found that Rb1 treatment reduced S.aureus-induced oxidative stress via significantly(p < 0.05)suppressing the accumulation of malondialdehyde(MDA)contents,and increasing the antioxidant enzyme activities of superoxide dismutase 1(SOD1),catalase(CAT),and glutathione peroxidase 1(Gpx1).Similarly,Rb1 markedly increased(p < 0.05)messenger RNA(m RNA)expression of antioxidant genes(SOD1,CAT and Gpx1)in comparison with S.aureus-stimulated RAW264.7 cells.Furthermore,Rb1 enhanced the antioxidant defense system through activating the Nrf2 signaling pathway.These findings showed that Rb1 treated group significantly(p < 0.05)up-regulated m RNA and protein expression of Nrf2 and its downstream associated genes were down-regulated in S.aureus-stimulated RAW264.7 cells.Moreover,a significant(p < 0.05)increase of both m RNA(Bax,caspase-3,caspase-9,cytochrome c and p53)and protein expression(Bax,caspase-3)of mitochondrial-apoptosis-related genes was observed,while the decrease of Bcl-2 in S.aureus-stimulated RAW264.7 cells was also observed.In addition,Rb1 therapy significantly(p < 0.05)reversed the m RNA and protein expression of these mitochondrial-apoptosis-related genes,as compared to the S.aureus-stimulated RAW264.7 cells.Taken together,Rb1 alleviated S.aureus-induced inflammation,oxidative stress and apoptosis in RAW264.7 cells.3.Ginsenoside Rb1 Ameliorates S.aureus-induced Inflammation,Oxidative stress and Apoptosis in lung tissue of miceThe aims of this study was to explore the anti-inflammatory,anti-oxidant and antiapoptotic effects of Ginsenoside Rb1 in S.aureus-induced ALI in mice.A total of 50 Kunming,6-8 weeks of age were randomly divided into five groups with ten mice in each group;as the control group,S.aureus group,S.aureus+Rb1(10 mg/kg)group,S.aureus+Rb1(20 mg/kg)group and Rb1 group(20 mg/kg).As compared with the control group,the results of physical morphology,histopathology,and lungs wet-to-dry weight ratio indicated that swelling,edematous lesions,hyperemia,hemorrhages,accumulation of inflammatory cells in interstitial spaces have been observed in S.aureus group,and was attenuated significantly(p < 0.05)with the administration of Rb1.Moreover,Rb1 significantly(p < 0.05)attenuated the production of myeloperoxidase(MPO).It was found that Rb1 treatment reduced S.aureus-induced oxidative stress via significantly(p < 0.05)suppressing the accumulation of malondialdehyde(MDA)and increased the antioxidant enzyme activities of superoxide dismutase 1(SOD1),Catalase(CAT),and glutathione peroxidase 1(Gpx1).Moreover,the results of RT-q PCR indicated that Rb1 significantly(p < 0.05)repressed IL-1β,IL-6,and TNF-α production in mice stimulated by S.aureus.Similarly,Rb1 markedly increased(p < 0.05)m RNA expression of antioxidant genes(SOD1,CAT and Gpx1)in comparison with S.aureus-stimulated group.The expression of NF-k B and MAPK signaling proteins determined by western blot assay,indicated that Rb1 has attenuated significantly(p < 0.05)the phosphorylation of P65,IKB-α JNK as well as ERK.The 20 mg/kg Rb1 dose group was used to explore protective effect of Rb1 on the oxidative stress and apoptosis.Likewise,Rb1 modulated the antioxidant defense system via activating the Nrf2 signaling pathway.These results depicted that Rb1 treated group significantly(p < 0.05)up-regulated m RNA and protein expression of Nrf2 and its downstream associated genes,which were down-regulated S.aureus-stimulated groups.Moreover,S.aureus significantly(p < 0.05)increased both m RNA(Bax,caspase-3,caspase-9,cytochrome c and p53)and protein expression(Bax,caspase-3)of mitochondrial-apoptosis-related genes.Likewise,the decrease of Bcl-2 in S.aureus-stimulated lung tissue of mice was also observed.Additionally,Rb1 therapy significantly(p < 0.05)reversed the m RNA and protein expression of these mitochondrial-apoptosis-related genes,as compared to the S.aureus-stimulated mice group.Cumulatively,Rb1 attenuated S.aureus-induced inflammation,oxidative stress and apoptosis in mice model of ALI.In summary,our experiments come to the following conclusions:(1)The pathogenesis of S.aureus-induced ALI is mainly due to,I.Activation of TLR2-mediated NF-k B and MAPK signaling pathway.II.Suppression of antioxidant enzymes and Nrf2 pathway.III.Activation of mitochondria-mediated apoptosis.(2)Rb1 played the role of anti-inflammatory,anti-oxidant,and anti-apoptosis in S.aureus-induced ALI:I.Rb1 displayed a protective anti-inflammatory effect in S.aureus-induced inflammation.The potential underlying mechanisms appear to be involved with the Rb1 in the suppression of TLR2-mediated NF-k B and MAPK signaling pathways.II.Rb1 displayed the protective anti-oxidant effects via antioxidant enzymes and Nrf2 in S.aureus-induced oxidative stress.Additionally,Rb1 attenuated S.aureusinduced oxidative damage via modulating the Nrf2 pathway.III.Rb1 displayed the protective anti-apoptotic effects via mitochondria-mediated apoptosis pathway in S.aureus-induced apoptosis.Moreover,Rb1 alleviated S.aureusinduced apoptosis through mitochondrial signaling pathway. |
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