The Role Of The NLRP3 Inflammasome In Heat Stress Damage

Part 1 Studies on the acute heat stress damageObjective: To study the damage of cells and tissues from the acute heat stress by in vitro and in vivo experiments.Methods:1.In vitro.(1)Effects of heat stress on cell damage: Murine peritoneal macrophages of C57BL/6 mice,as the target cells,were put into 6-well plates to culture.Murine peritoneal macrophages of C57BL/6 mice were divided into 4 groups: blank control group(37 ?),heat stress(38 ?)with lipopolysaccharides(LPS)group,heat stress(40 ?)with LPS group and heat stress(42 ?)with LPS group.The three heat stress group were heated in different time point(1 hour,2 hours,4 hours).Blank control group(37 ?)was cultured in temperature of 37 ? for 1 hour,2 hours and 4 hours.Cells of the heat stress groups were added LPS for priming 6 hours and then give different temperatures of heat stress for 1 hour,2 hours and 4 hours.After the treatment,we observed the cellular morphology by microscope,detected the survival rate of cells by the method of MTT,and the release of cellular lactate dehydrogenase(LDH).(2)The release of inflammatory cytokines after heat stress: Murine peritoneal macrophages of C57BL/6 mice were divided into 4 groups: blank control group,heat stress group,LPS group and heat stress with LPS group.LPS group and heat stress with LPS group were added LPS for priming 6 hours and then give different temperatures of heat stress: 38 ?(1 hour,2 hours,4 hours),40 ?(1 hour,2 hours,4 hours),and 42 ?(1 hour,2 hours,4 hours).The supernatants of the cell culture fluid at respective temperature and time were accumulated.Then detect the concentration of IL-1?,IL-6 and TNF-?,using the method of ELISA.2.In vivo.(1)C57BL/6 mice were randomly divided into four groups: control group and heat stress(38 ?,40 ? and 42 ?)group with LPS.Mice of the heat stress group with LPS were injected with LPS(1mg/kg)in advance for 4 hours,then exposed to heat stress of different temperature.The rectal temperatures of mice were measured at the beginning and at 2 hours(or dying)after the heat stress.The survival mice were killed at 2 hours after the heat stress for observing the release of IL-1?.(2)C57BL/6 mice were randomly divided into two groups: control group and LPS group.Mice of the LPS group were intraperitoneal injected with LPS(1mg/kg)in advance for 4 hours,then checked the hemogram indexs.(3)C57BL / 6 mice were divided into blank control group,heat stress group,LPS group and heat stress with LPS group of 40 ?.After being heated,the anal temperature changes of four groups of mice were monitored every 15 min,and the survival time was recorded,and made the survival curve.Results: 1.In vitro.(1)Effects of heat stress on cell damage: After 4 hours heat stress of 42?,obvious changes were observed morphologically in cells shrinking,volumes decreasing,intercellular space increasing,and contours of cell membrane becoming blur,and half of cells being dead.Few cells died in the groups of 40 ?,4 hours and 42 ?,2 hours.And there is no significant change in other groups.Results of MTT testing showed that cells survival rate of the group 42 ?,4 h decreases significantly after the heat stress,while the survival rate of other groups had no significant change.LDH testing results showed that,compared to group of 37 ?(1 h,2 h,4 h),the release level of LDH increased depending on the increase of the temperature and the extension of time.(2)The release of inflammatory cytokines after heat stress: Results of IL-1?,IL-6 and TNF-? testing showed that there was a significant change in LPS group and heat stress with LPS group.2.In vivo.(1)The rectal temperature of group in 42 ? was the highest after 2 hours of heat stress.The group 40 ? was a bit higher than the group 38?.The level of IL-1? in serum of mice of group 40 ? were higher than the others.(2)After the treatment of LPS(1 mg/kg)for 4 h,the blood of mice in WBC and % NEUT compared with the blank control group were significantly increased,and % LYMPH was significantly decreased.(3)By monitoring the rectal temperature of control group,LPS group,heat stress and LPS composite 40 ? temperature heat stress mice,we found that there was no obvious change of rectal temperature in blank control group and LPS group.Rectal temperature of heat stress group was flat elevated.After 15 min,the rectal temperature of heat stress with LPS group was increased rapidly than heat stress group.Survival analysis showed that LPS group of wild type mice in 4 h observation period there was no death.Median survival time of heat stress group was 2.72 h,the average survival time was 2.56 h;LPS composite heat stress group of wild type mice survival time was significantly shortened and the median survival time was 1.47 h,average survival time was 1.47 h.Conclusions: Heat stress could result in different degrees of damage to cells and mice,and the degree of the damage depends on the temperature and duration of the heat stress.Pre-existing LPS reduced the body's tolerance to heat,and the mice were prone to heat attack,and the survival time was shortened.Part 2 Heat stress enhances LPS-induced NLRP3 inflammasome activationObjective: To study whether the heat stress could activate NLRP3 inflammasome.Methods: 1.In vitro.(1)According to the experiments in Part 1,heat stress of 40 ?,4h was a suitable condition for the study of the mechanisms of the NLRP3 inflammsome activation,with highest secretion of IL-1? and lowest damage of cells.Murine peritoneal macrophages of C57BL/6 mice were divided into 4 groups: blank control group,heat stress group,LPS group and heat stress with LPS group.The supernatants of the cell culture fluid at respective temperature and time were accumulated.Then detect the concentration of IL-1?,the main inflammatory factor released by NLRP3 inflammasome activation,using the method of ELISA,and the expression of inflammsome related proteins with Western Blot,including NLRP3,pro-Caspase-1,ASC,p10 and pro-IL-1?.(2)Cell total RNA of all groups were extracted,and using Real-time PCR to assay the expression changes of NLRP3?ASC and Caspase-1.2.In vivo.(1)C57BL/6 mice were separated into 4 groups: blank control,heat stress group,LPS group and heat stress group with LPS.Mice in the heat stress group were placed in a high temperature simulation cabin(humidity: 60 % ± 5 %)with the temperature of 40?.Mice in the LPS group are injected with LPS.Mice in the heat stress with LPS group are injected with LPS priming for 4 hours,and then placed in thehigh temperature simulation cabin(humidity: 60 % ± 5 %)with the temperature of 40?.Set the time point of mice dead in the heat stress with LPS as the end.The concentration of IL-1? in mice blood serum was measured using the method of ELISA.(2)Hepatic tissue of these mice were collected,from which the total proteins were extracted for detection with Western Blot.Results: 1.In vitro.(1)the concentrations of IL-1? in the supernatant of the cell culture fluid at respective temperature and time tested by ELISA method showed that the concentration of IL-1? in the group of heat stress with LPS(40 ?,4 h)had a significant increase compared to other groups(P<0.05).The results of Western Blot testing showed that protein expression levels of NLRP3?pro-Caspase-1?ASC?p10 and pro-IL-1? have an increase in the group of heat stress with LPS.(2)The results of RT-PCR testing showed that gene expression levels of Nlrp3?Asc and Caspase-1 had an increase in the group of heat stress with LPS compared to others(P<0.05).This part of results shows that heat stress of 40 ?,4 h with LPS priming could activate the NLRP3 inflammsome.2.In vivo.(1)The concentration of IL-1? in mice serum of the group heat stress with LPS had a significant increase compared to other 3 groups(P<0.05).(2)Western Blot testing showed that heat stress combined with LPS could promote the expression of NLRP3,pro-Caspase-1,pro-IL-1?and IL-1? in hepatic tissue.(3)Western Blot testing showed that heat stress combined with LPS could promote the expression of NLRP3,IL-1? and p10 in hypothalamus.Conclusions: Cell experiments showed that heat stress with LPS could activate the NLRP3 inflammasome in Murine peritoneal macrophages of C57BL/6 mice,and facilitate the expression of NLRP3,pro-Caspase-1,ASC,p10,pro-IL-1? and the release of IL-1?.Animal experiments showed that heat stress with LPS could promote the release of IL-1? in mice serum and the protein expression of NLRP3,pro-Caspase-1,pro-IL-1? and IL-1? in hepatic tissue,also the protein expression of NLRP3,IL-1? and p10 in hypothalamus.These results showed that heat stress with LPS could activate NLRP3 inflammsome in vitro and in vivo.Part 3 Effects of the NLRP3 inflammasome on acute heat stress damageObjective:Expore the function of the NLRP3 inflammasome in acute heat stress damage.Methods: Build the cell model and animal model of heat stress damage respectively,using the mice of 3 gene knocked-out related to the NLRP3 inflammasome(Nlrp3-/-,Caspase-1-/-,Asc-/-).1.In vitro.Target cells were murine peritoneal macrophages of C57BL/6,Nlrp3-/-,Caspase-1-/-and Asc-/-mice and heat stress condition was 40?,4 h.Target cells were separated into 4 groups: blank control,heat stress group,LPS group and heat stress with LPS group.We accumulated the supernatant of the cell culture fluid at respective temperature and time,using the method of MTT for the measurement of cell survival rate,ELISA forthe concentration of LDH and IL-1? releasement.2.In vivo.(1)Several groups were needed: blank control(C57BL/6 WT),heat stress group(C57BL/6 WT),LPS group(C57BL/6 WT),heat stress group with LPS(C57BL/6 WT),heat stress group with LPS(Nlrp3-/-),heat stress group with LPS(Caspase-1-/-)and heat stress group with LPS(Asc-/-).Mice in the heat stress group were placed in a high temperature simulation cabin with the temperature of 40 ?.Mice in the heat stress group with LPS were injected with LPS priming for 4 hours,placed in a high temperature simulation cabin with the temperature of 40 ?.Then did the following analysis: 1)Prepared the blood serum when mice dead and measured the concentration of IL-1? by ELISA.2)After being heated,the anal temperature changes of four groups of mice were monitored every 15 min.3)Recorded the time when mouse dead,and make the survival curves.(2)C57BL/6 mice were separated into 4 groups: heat stress group,heat stress group with LPS,heat stress group with LPS + 1 ?g IL-1? neutralizing antibody(IL-1? NA)and heat stress group with LPS + 5 ?g IL-1? NA.Then placed the mice in a high temperature simulation cabin with the temperature of 40 ?.Then did the following analysis: 1)After being heated,the anal temperature changes of four groups of mice were monitored every 15 min.2)Recorded the time when mouse dead,and make the survival curves.Results: 1.In vitro:(1)The method of MTT at respective temperature and time showed that the survival rate of the group of heat stress with LPS(C57BL/6 wild)was a bit lower than the others.(2)The concentrations of LDH in the supernatant of the cell culture fluid of the group of heat stress with LPS(C57BL/6 wild)was a bit higher than the others(3)The concentrations of IL-1? in the supernatant of the cell culture fluid at respective temperature and time tested by ELISA method showed: the concentration of IL-1? in the group of heat stress with LPS(C57BL/6 wild)had a significant increase compared to other temperature and time(P<0.05).2.In vivo.(1)the testing of the concentration of IL-1? in the blood serum by ELISA method shows that the concentration of IL-1? in the group of heat stress with LPS has a significant increase compared to other 3 groups(P<0.05).(2)The rectal temperature of three groups of genes knocked out mice's monitoring results show that Nlrp3-/-,Caspase-1-/-and Asc-/-mice's rectal temperature were lower than the wild type heat stress with LPS group in the same monitoring point.(3)The survival analysis showed that after the treatment of heat stress with LPS,the median survival time of mice with Nlrp3-/-,Caspase-1-/-and Asc-/-were longer than wild type mice(P<0.05 or P<0.01).(4)After the treatment of IL-1? NA,the same time monitoring of mice's rectal temperature were lower than no IL-1? NA heat stress with LPS group mice,and heat stress with 5 ?g IL-1? NA mice's rectal temperature were lower than 1 ?g IL-1? NA group.(5)After 5 ?g IL-1? NA,the survival time of heat stress with LPS mice was significantly prolonged.Conclusion: Cell experiments and animal experiments showed that the NLRP3 inflammasome activation is related with the acute heat stress damage.Inhibition of NLRP3 inflammasome can prolong the survival time of mice with heat stress and LPS,which is related to inhibiting IL-1?.