The Effects And Mechanisms Of ?-glucan From Saccharomyces Cerevisiae On Oxidative Stress,inflammation And Apoptosis In RAW264.7 Cells

Macrophages are highly plastic cells of the innate immune system.They participate in a variety of pathological processes and are highly resistant to oxidative stress,inflammation and apoptosis induced by many toxic stimuli.?-glucan from Saccharomyces cerevisiae has been proved to be an effective immune enhancer.Furthermore,our group confirmed that ?-glucan can reduce oxidative stress in meat sheep.This study firstly investigated that the antioxidant and anti-inflammatory effects of?-glucan through using LPS-induced RAW264.7 cells as oxidative stress and inflammatory response models.Then,based on the oxidative stress model,our aimed is to explore the molecular mechanism of anti-oxidation,anti-inflammatory and anti-apoptotic effects of ?-glucan on RAW264.7 cells.It will provide new ideas for the prevention and treatment of oxidative stress-related diseases.In this study,the mechanism of antioxidant,anti-inflammatory and anti-apoptotic action of ?-glucan from Saccharomyces cerevisiae was studied from the aspect of cell signaling pathway.The results are as follows:(1)In the first experiment,LPS was used as an inducer,oxidative stress and pro-inflammatory cytokines as indicators,for establishing oxidative stress and inflammation models,and the effects of LPS on the oxidative damage of RAW264.7 cells were explored.The results showed that the production of oxidative stress indicators MDA and ROS increased significantly,and the enzyme activities of antioxidant indicators SOD,CAT and GSH-Px significantly decreased in RAW264.7 cells treated by 0.1?5 ?g/m L LPS for 24 h;the release of pro-inflammatory cytokines IL-1?,IL-6,and TNF-? in cell culture supernatant and content of COX-2 and i NOS in cell were significantly increased.With reference to multiple indicators,1 ?g/m L LPS was used as the optimal concentration to establish a cell oxidative damage model.(2)Based on the first experiment,the purpose of the second experiment was to explore the protective effect of ?-glucan on oxidative stress and inflammatory damage in LPS-induced RAW264.7 cells.The results showed that ?-glucan enhanced the activities of antioxidant enzymes SOD,CAT,GSH-Px and HO,and inhibited the production of MDA and ROS induced by LPS to exert antioxidant effects;?-glucan plays an anti-inflammatory effect by inhibiting the release of IL-1?,IL-6,TNF-?,COX-2,and i NOS.It is speculated that ?-glucan activates the antioxidant transcription factor to play a pre-protective effect.These results fully demonstrated that ?-glucan has dual regulatory effects in antioxidant and antiinflammatory properties,thereby inhibiting cell apoptosis and ROS production,and then playing a protective effect on RAW264.7 cells.However,the signal transduction mechanism in regluating antioxidation and antiinflammatory needs to be further verified in the regression oxidative stress model.(3)Based on the second experiment,the third experiment aimed to explore the molecular mechanism of ?-glucan regulating LPS-induced oxidative stress in RAW264.7cells.In order to clarify the antioxidant molecular mechanism of ?-glucan,we designed the experiment adding ?-glucan alone,and the results showed that ?-glucan could activate the receptor Dectin-1 mediated Nrf2/HO-1 signal transduction pathway.In the oxidative stress model,the results showed that LPS did not activate the expression of Dectin-1 but inhibited the expression of Nrf2 and HO-1,?-glucan could significantly up-regulate the expression of Dectin-1,Nrf2 and HO-1 induced by LPS,inhibit ROS production,but these effects of ?-glucan could be reversed by Sn PP.These results indicated that ?-glucan could inhibit LPS-induced oxidative stress in RAW264.7 cells through the Dectin-1/Nrf2/HO-1 signaling pathway.(4)On the basis of third experiment,the fourth experiment aimed to explore the molecular mechanism of ?-glucan in regulating the inflammatory response in LPS-induced RAW264.7 cells through the Nrf2/HO-1 signaling pathway.The results showed that in the inflammation model,LPS significantly activated the expression of the NF-?Bp65,resulting in a significant increase in the expression of IL-1?,IL-6,TNF-?,COX-2 and i NOS.Moreover,?-glucan could significantly inhibited the expression of NF-?Bp65 and its downstream pro-inflammatory cytokines induced by LPS,while this anti-inflammatory effect could be reversed by Sn PP.These results indicated that ?-glucan could inhibit the inflammatory response through the Nrf2/HO-1 signaling pathway in LPS-induced RAW264.7 cells.(5)Based on the results of fourth experiment,the fifth experiment aimed to explore the molecular mechanism of ?-glucan regulating LPS-induced NLRP3 formation in RAW264.7 cells through the Nrf2/HO-1 signaling pathway.The results showed that LPS significantly activated the NLRP3 inflammasome,enhanced the expression of ASC,IL-18 and Caspase-1,and after the addition of ?-glucan,it could significantly inhibit the expression of NLRP3,ASC,Caspase-1 and IL-18,but this inhibitory state could be reversed by Sn PP.These results indicated that ?-glucan could inhibit NLRP3 formation in LPS-induced RAW264.7 cells through Nrf2/HO-1 signaling pathway.(6)Relying on the above experiments and oxidative stress can cause cell apoptosis,the sixth experiment aimd to explore the molecular mechanism of ?-glucan regulating apoptosis in LPS-induced RAW264.7 cells through the Nrf2/HO-1 signaling pathway.The results showed that LPS significantly enhanced the rate of apoptosis,and ?-glucan significantly reduced the rate of apoptosis;LPS significantly enhanced the expression of Bax but inhibited that of Bcl-2;after the addition of ?-glucan,the expression of Bax decreased significantly,and the Bcl-2 expression increased significantly,but these effects could be reversed by Sn PP.These results indicated that ?-glucan could inhibit apoptosis in LPS-induced RAW264.7 cells through the Nrf2/HO-1 signaling pathway.In summary,this study clarifies that ?-glucan can activate the Dectin-1 mediated Nrf2/HO-1 signaling pathway to inhibit ROS production,NF-?Bp65 and NLRP3 expression and apoptosis in RAW264.7 cells,thereby play protective role.Therefore,activated the Nrf2/HO-1 signaling pathway as a target is expected to become a new way to prevent and treat oxidative stress-related diseases.