Effect And Mechanism Of Hydrogen On Hippocampal Microglia Polarization In Septic Mice

Sepsis is one of the common causes of intensive care unit admitting,which have threaten public health seriously and caused a great burden to the society and family.Septic patients are often attacked by sepsis associated encephalopathy,presented as diffused brain dysfunction,which affect their prognosis and quality of life seriously.The mechanism of sepsis associated encephalopathy have not been fully elucidated,and there is no effective treatment in clinical practise.Previous researches from our team found that hydrogen inhalation could attenuate cerebral inflammation and sepsis associated encephalopathy.Its mechanism,however,still needs further investigation.As the macrophage in cerebral,microglia have received much more attention recently.Microglia consists of two types of polarization and play different role in cerebral inflammation.Whether hydrogen alleviates sepsis associated encephalopathy by regulating microglia polarization remained unknown.In this research,we performed both vivo and vitro researches to investigate the effect of hydrogen on cerebral inflammatory reaction,microglial polarization and investigated the mechanism of these changes through molecular biological techniques.All these efforts aimed to provide experimental and theoretical basis for the application of hydrogen in clinical practice.PART 1.Effects of molecular hydrogen inhalation on microglial polarization in septic miceObjective: To explore the effect of molecular hydrogen inhalation on microglial polarization and central inflammation in septic mice.Methods: Mice were divided randomly into four independent groups: sham operation group(Sham group),sham operation plus hydrogen inhalation group(Sham+H group),cecum ligation and perforation group(CLP group)and cecum ligation and perforation plus hydrogen inhalation group(CLP+H group).Septic model was performed by caecum ligation and perforation(CLP operation),2% hydrogen was inhaled for 1h at 1h and 6h after operation.The survival rates of mice were observed for 8 days after operation.The changes of learning and memory function of mice were detected by radial-arm maze and fresh object recognition experiment.ELISA were used to detect inflammatory factor infiltration in hippocampus of mice.Microglia M1/M2 polarizational changes from hippocampus were observed by immunofluorescence and Western blotting.Apoptosis in hippocampal sections were also observed.Results: Compared with group Sham,there were more working memory errors,reference memory errors,less new objects exploration time and decreased preference index in group CLP.Group CLP also exhibited decreased survival rates,increased IL-1β,TNF-α,HMGB1,IL-4,IGF-1 and TGF-β in hippocampus.Microglia of M1 polarization were dominated and more hippocampal apoptosis were observed.Hydrogen inhalation could alleviated the above symptoms.As compared with group CLP,group CLP+H exhibited increased survival rates,less errors on working memory and reference memory,more new objects exploration time and increased preference index.The amounts of IL-1β,TNF-α and HMGB1 decreased while IGF-1,TGF-β and IL-4 increased in hippocampus.Hydrogen inhalation also depressed M1 polarization and promoted M2 polarization,associated with less hippocampal apoptosis.Conclution: Hydrogen inhalation depressed M1 polarization while promoted M2,alleviated the cerebral inflammation and hippocampal apoptosis,improved learning and memory function and survival rates in septic mice.PART 2.Effects of hydrogen on LPS induced microglia polarization and its mechanismObjective: To testify the direct regulation from hydrogen on LPS induces microglia polarization and investigate its mechanism.Methods: This section included three sub-experiments.First,BV-2 microglia were cultured in vitro and M1 polarization was induced by LPS.Hydrogen-rich medium was prepared and microglia were incubated with it to simulate hydrogen treatment in vitro for 24 h.The inflammatory factors in the supernatant were measured by ELISA.The markers of microglia M1/M2 polarization were detected by q PCR and flow cytometry.Microglia were also co-cultured with HT-22 neurons through transwell techniques for another 24 h and neuronal viability and apoptosis were detected.Second,we detected ·OH,ONOO-,superoxide dismutase(SOD),glutathione(GSH) and DNA damage in microglia treated with LPS and hydrogen.The changes of PARP-1,Sirt-1 and NAD+ were also detected.Meanwhile,we further confirmed the relationship between PARP-1 and Sirt-1 in LPS induced microglia by the application of PARP-1 siRNA and its inhibitor 3-AB.Third,we verified whether hydrogen regulated microglia polarization through Sirt-1 activation by the application of Sirt-1siRNA and its inhibitor EX-527.Results: First,we discovered that LPS incubation could promoted TNF-α,IL-1β,HMGB1,IL-4,IGF-1and TGF-β from the supernatant,with increased M1 markers transcription of i NOS,MCP-1 and increased Iba-1+CD86+microglia,co-cultured neurons exhibited decreased viability and increased apoptosis.Hydrogen-rich medium incubation reduced TNF-α,IL-1β and HMGB1 while enhanced IL-4,IGF-1and TGF-β from the supernatant.i NOS and MCP-1 transcription and Iba-1+CD86+microglia decreased whereas Arg-1 and Ym-1 transcription and Iba-1+CD206+microglia increased.Co-cultured neurons exhibited improved viability and decreased apoptosis.Second,we discoveded that LPS incubation promoted ·OH,ONOO-,DNA damage,PARP-1 expression and activity whereas reduced NAD+,SOD,GSH,Sirt-1expression and activity.Hydrogen-rich medium incubation alleviated the situation with decreased ·OH,ONOO-,DNA damage,PARP-1 activity and expression while increased NAD+,SOD,GSH,Sirt-1 activity and expression.The application of PARP-1 siRNA or 3-AB to LPS induced microglia promoted Sirt-1 activity and expression as well.Third,the polarizational regulation of hydrogen-rich medium were eliminated by Sirt-1siRNA or EX-527,presented as increased i NOS and MCP-1transcription and decreased Arg-1 and Ym-1 transcription.Conclusion: Hydrogen reduced LPS induced oxygen radical promotion,alleviated DNA oxidative stress damage,inhibited PARP-1 over-activation,promoted Sirt-1expression and activity,decreased microglia M1 polarization and promoted M2 polarization.PART 3.The downstream mechanism of polarizational regulation of hydrogen on LPS induced microgliaObjective: To investigate the downstream mechanism of M1 inhibition and M2 promotion of Sirt-1 on microglia exerted by hydrogen.Methods: This section included three sub-experiments.First,microglia M1 polarization were performed by LPS,then incubated by hydrogen-rich medium and Sirt-1siRNA were used to knock down Sirt-1 expression.The expression of Sirt-1,p-IκBα,cytoplasmic ac-p65,p-p65 and nuclear ac-p65,p-p65,p65 were detected by Western Blotting.TNF-α,IL-1β,i NOS and MCP-1 m RNA transcription were detected by q PCR.Second,microglia M2 polarization were performed by IL-4 and confirmed by q PCR detection of TGF-β,IL-10,Arg-1 and Ym-1.Western Blotting were used to detect the expression of Sirt-1,p-AMPK,p-m TOR,LC3Ⅱ/Ⅰ,Beclin-1and p62.Sirt-1siRNA and autophagy inhibitor 3-MA were used to testify whether Sirt-1/AMPK/autophagy passway participated in regulating M2 polarization.Third,microglia M1 polarization were performed by LPS,incubated by hydrogen-rich medium and 3-MA was used to verify whether hydrogen promoted M2 polarization through Sirt-1/AMPK/autophagy passway.Results: First,we discovered that increased p-IκBα,cytoplasmic ac-p65,p-p65,nuclear ac-p65,p-p65 and p65 after LPS incubation.Transcription of M1 markers such as TNF-α,IL-1β,i NOS and MCP-1 increased.Hydrogen-rich medium incubation reduced p-IκBα,cytoplasmic ac-p65,p-p65,nuclear ac-p65,p-p65 and p65.Microglia M1 markers transcription were also down-regulated.The above effects of hydrogen-rich medium were attenuated by Sirt-1siRNA.Second,IL-4successfully induced M2 polarization with increased TGF-β,IL-10,Arg-1 and Ym-1transcription.p-AMPK,LC3Ⅱ/Ⅰand Beclin-1were also up-regulated while p-m TOR and p62 were reduced.The above effects of IL-4 were attenuated by Sirt-1siRNA and3-MA.Third,hydrogen-rich medium incubation up-regulated Sirt-1,p-AMPK,LC3Ⅱ / Ⅰ and Beclin-1 expression while decrease p62 expression.The use of 3-MA attenuates M2 promotion by hydrogen-rich medium on microglia.Conclusion: Hydrogen promoted p65 deacetylation through Sirt-1 activation,reduced its nucleation and inhibited microglia M1 polarization.Hydrogen promoted microglia M2 polarization through activating Sirt-1/AMKP/ autophagy pathway.Summary:1.Hydrogen inhalation regulated microglia polarization from M1 towards M2 in septic mice,accompanied with alleviated cerebral inflammation,hippocampal apoptosis,improved learning and memory function and increased survival rate.2.Hydrogen regulated LPS induced microglia polarization from M1 towards M2 and attenuated microglia inflammatory reaction in vitro.The mechanism is related to the reduction of oxygen radicals and oxidative stress on DNA,inhibited PARP-1over-activation,and up-regulated Sirt-1 activity and expression.3.Hydrogen promoted NF-κB-p65 deacetylation through Sirt-1 activation,reduced its nucleation and inhibited microglia M1 polarization.Hydrogen promoted microglia M2 polarization through activating Sirt-1/AMKP/ autophagy pathway.