The Role Of SIRT1-Mediated Deacetylation In Postoperative Cognitive Dysfunction In Aged Rats

Part Ⅰ Anesthesia and surgery aggravates the age-related decrease in SIRT1 expression and induces neuroinflammation in the hippocampus of aged ratsObjective: To detect the effect of anesthesia and surgery on SIRT1 expression and neuroinflammation in the hippocampus of aged rats.Methods: Collect the hippocampus tissue of Sprague-Dawley rats(postnatal day 7,P7;postnatal day 14,P14;postnatal day 57,P57;and 21 months old,M21)and evaluate the levels of SIRT1 in the hippocampus of four different groups using Western Blot analysis.The 21-months-old male Sprague–Dawley rats were randomly assigned to the following groups: CON group,the control group rats were treated with control gas;ISO+SUR group,the anesthesia and surgery group rats received splenectomy under 2% isoflurane anesthesia.The hippocampus of two groups were harvested 24 h after treatment.The expression of SIRT1,DNMT1,ac-NF-κB,IL-6 in the hippocampus were evaluated using Western Blot analysis(n=6).The expression of ac-tau(k280),actau(k686),p-tau(AT8)were evaluated using Western Blot analysis(n=6).On postoperative hour 24,brains were harvested and fixed in 4% paraformaldehyde.Tenmicron-thick frozen sections were cut from the rat brains.Microglia activation was detected using immunostaining.Results:(1)Western Blot analysis showed that,among four different aged rats,21-months-old rats showed significantly decreased level of SIRT1 in the hippocampus compared with other groups(P<0.05).(2)In 21-months-old male rats,Western Blot analysis showed that,anesthesia and surgery significantly decreased the expression of SIRT1 and DNMT1(P<0.05),and increased the expression of ac-NF-κB(P<0.05),IL-6(P<0.001)in the hippocampus compared with rats in CON group.(3)compared with CON group,anesthesia and surgery increased the expression of ac-tau(k280),actau(k686),p-tau(AT8)in the hippocampus(P<0.001).(4)The immunostaining data showed that anesthesia and surgery increased Iba1 positive cells in CA1,CA3 and DG regions of hippocampus compared with rats in CON group.Conclusion: Anesthesia and surgery aggravates the age-related downregulation of SIRT1 expression in the hippocampus.Anesthesia and surgery might induce neuroinflammation with downregulation of DNMT1 expression,upregulation of acNF-κB,IL-6 expression,and over-activation of microglia in the hippocampus of aged rats.Anesthesia and surgery induce upregulated tau acetylation and phosphorylation.Part Ⅱ Activation of SIRT1 inhibits LPS-induced BV2 cell over-activation and decreased levels of tau acetylation and phosphorylation in primary neuronsObjective: To detect the role of SIRT1 in the process of BV2 cell activation and tau acetylation and phosphorylation in primary neurons.Methods: The BV2 cell populations were allocated into the following groups: CON group,the control group cells were treated with 0.1% DMSO for 24 h;LPS group,the LPS treatment group cells were treated with LPS(100ng/ml)and 0.1% DMSO for 24 h;RES group,the resveratrol treatment group cells were treated with resveratrol(1μmol)and 0.1% DMSO for 24 h;RES+LPS group,the resveratrol+LPS treatment group cells were treated with resveratrol(1 μmol),LPS(100 ng/ml)and 0.1% DMSO for 24 h.After treatment,conditional medium(CM)were harvested,centrifuged and filtered.CMs were stored at-80℃ until use.Collect the BV2 cells of four groups to detect the expression of SIRT1,ac-NF-κB,IL-6 using Western Blot(n=6).Primary neurons were prepared from the hippocampus and cortex of postnatal 24 h rat and allocated into the following groups: CON CM group: the cells were treated with CON CM for 24 h;LPS CM group: the cells were treated with LPS CM for 24 h;RES CM group: the cells were treated with RES CM for 24 h;RES+LPS CM group: the cells were treated with RES+LPS CM for 24 h.After treatment,the primary neurons were collected to detect the expression of SIRT1,ac-tau(k280),ac-tau(k686)and p-tau(AT8)(n=6).Results:(1)In BV2 cells,the Western Blot analysis showed that LPS can decrease the expression of SIRT1(P<0.001),increase the expression of ac-NF-κB,IL-6 in BV2 cells compared with the CON group(P<0.001).Compared with LPS group,RES+LPS group showed increased expression of SIRT1(P<0.05)and decreased expression of ac-NF-κB(P<0.05),IL-6(P<0.001)in BV2 cells;(2)In primary neurons,the Western Blot analysis showed that LPS CM treatment decreased the expression of SIRT1 and increased the expression of ac-tau(k280),ac-tau(k686)and p-tau(AT8)compared with CON CM group(P<0.001).Compared with LPS CM group,RES+LPS CM group showed increased expression of SIRT1(P<0.001),and decreased expression of ac-tau(k280),ac-tau(k686)and p-tau(AT8)(P<0.05).Conclusion: Activation of SIRT1 can attenuate LPS-induced SIRT1 downregulation and over-activation in BV2 cell lines,and decrease the levels of tau acetylation and phosphorylation in primary neurons.Part Ⅲ Activation of SIRT1 inhibites neuroinflammation,tau acetylation and cognitive impairment in aged rats exposed to anesthesia and surgeryObjective: To detect the role of SIRT1 in anesthesia and surgery-induced neuroinflammation,tau acetylation and cognitive dysfunction in aged rats.Methods: Experiment 1: The 21-months-old male Sprague–Dawley rats(weighting 620 g ~ 670 g at the start of the experiment)were randomly assigned to the following groups: CON group,the control group rats were injected with the DMSO-saline solution for 7 d and treated with control gas for 2 h;ISO+SUR group,the anesthesia and surgery group rats were injected with the DMSO-saline solution for 7 d and followed by splenectomy under 2% isoflurane anesthesia for 2 h;RES group,the resveratrol group rats were injected with resveratrol(10 mg/kg)for 7 d and treated with control gas for 2 h;RES+ISO+SUR group,the resveratrol and anesthesia and surgery group rats were injected with resveratrol(10 mg/kg)for 7 d and followed by splenectomy under 2% isoflurane anesthesia for 2 h.The hippocampus were harvested 24 h after anesthesia and surgery.The expression of SIRT1,DNMT1,ac-NF-κB,IL-6 in the hippocampus were evaluated using Western Blot analysis(n=6).The protein level of TNF-α and IL-1β in the hippocampus were examined by ELISA(n=6).The expression of CD86 m RNA,CD206 m RNA and SOCS3 m RNA in the hippocampus were examined by PCR(n=6).On postoperative hour 24,brains were harvested and fixed in 4% paraformaldehyde.Ten-micron-thick frozen sections were cut from the rat brains.Microglia activation was evaluated using immunostaining.The hippocampus were harvested 24 h after anesthesia and surgery,the expression of ac-tau(k280),actau(k686),p-tau(AT8)were evaluated using Western Blot analysis(n=6).Experiment 2: The 21-months-old male Sprague–Dawley rats(weighting 620 g ~ 670 g at the start of the experiment)were randomly assigned to the following groups: CON group,the control group rats were injected with the DMSO-saline solution for 7 d and treated with control gas for 2 h;ISO+SUR group,the anesthesia and surgery group rats were injected with the DMSO-saline solution for 7 d and followed by splenectomy under 2% isoflurane anesthesia for 2 h;RES group,the resveratrol group rats were injected with resveratrol(10 mg/kg)for 7 d and treated with control gas for 2 h;RES+ISO+SUR group,the resveratrol and anesthesia and surgery group rats were injected with resveratrol(10 mg/kg)for 7 d and followed by splenectomy under 2% isoflurane anesthesia for 2 h.At days 12-13 after anesthesia and surgery,the animals were subjected to fear conditioning test for evaluation of associated learning and memory(n=10).At days 15-20 after anesthesia and surgery,the animals were subjected to Morris Water Maze for evaluation of spatial learning and memory(n=10).Results:(1)Western Blot analysis showed that,compared with CON group,ISO+SUR group showed decreased levels of SIRT1 and DNMT1(P<0.05),increased levels of acNF-κB(P<0.05),IL-6(P<0.001)in the hippocampus;compared with ISO+SUR group,RES+ISO+SUR group showed increased trend of SIRT1 and DNMT1 levels(P<0.05),and decreased levels of ac-NF-κB(P<0.05),IL-6(P<0.001)in the hippocampus;(2)The immunostaining data showed that anesthesia and surgery increased Iba1 positive cells in CA1,CA3 and DG regions of hippocampus compared with the control group.Whereas compared with ISO+SUR group,RES+ISO+SUR group rats showed decreased Iba1 positive cells in CA1,CA3 and DG regions of hippocampus;(3)The PCR data showed that,compared with control group,anesthesia and surgery increased the expression of CD86(P<0.001),a marker of cytotoxic M1 phenotype of microglia,and SOCS3(P<0.001),a marker of immunomodulatory M2 b phenotype of microglia,while decreased the expression of CD206(P<0.001),a marker of repair/regeneration M2 a phenotype of microglia.Compared with ISO+SUR group,RES+ISO+SUR group showed decreased expression of CD86 and SOCS3(P<0.001),and increased expression of CD206(P<0.001);(4)The ELISA data showed that,compared with CON group,anesthesia and surgery increased the protein level of TNF-α and IL-1β in the hippocampus(P<0.001);compared with ISO+SUR group,RES+ISO+SUR group showed decreased expression of TNF-α and IL-1β(P<0.001)in the hippocampus;(5)Fear conditioning test showed that anesthesia and surgery decreased the freezing time in context test compared with control group(P<0.05),and resveratrol pretreatment increased the freezing time in context test compared with anesthesia and surgery group(P<0.05).Morris Water Maze test showed that anesthesia and surgery increased the escape latency on the fifth day of training(P<0.001),decreased the percentage of time spent in the target quadrant and the frequency of target zone transitions compared with control rats(P<0.05).Compared with ISO+SUR group,RES+ISO+SUR group showed decreased escape latency on the fifth day of training(P<0.05),increased trend in the percentage of time spent in the target quadrant and higher frequency of target zone transitions(P<0.05).(6)Western Blot analysis showed that,compared with CON group,ISO+SUR group showed increased levels of ac-tau(k280),ac-tau(k686)and p-tau(AT8)protein in the hippocampus(P<0.001);compared with ISO+SUR group,RES+ISO+SUR group showed decreased trend of ac-tau(k280),ac-tau(k686)and ptau(AT8)protein levels(P<0.05).Conclusion: Anesthesia and surgery can induce microglia over-activation,increase neurotoxic phenotype of microglia,upregulate inflammatory factors,upregulate tau acetylation and phosphorylation,and cause cognitive impairment in aged rats.Pretreatment with resveratrol can attenuate anesthesia and surgery-induced neuroinflammation and cognitive dysfunction.