The Possible Mechanism Of Honokiol Against Postoperative Cognitive Decline In Mice

Part Ⅰ The expression of NLRP3 inflammasome in the hippocampal tissue of mice with postoperative cognitive declineObjective To investigate the expression of NLRP3 inflammasome in hippocampus of mice with postoperative cognitive decline.Methods Thirty C57BL/6j mice were split into two groups: control group(group C)and surgery group(group S).Mice in S group received laparotomy surgery under sevoflurane anesthesia,mice in C group did not undergo surgery.On postoperative 1 and 3 days,fear conditioning test and open field test were used to test the cognitive function of mice in each group.Then their hippocampus were removed from brain to detect the levels of NLRP3,Caspase 1 and ASC protein.Results 1.Open field test showed that there was no significant change in the total distance between group C and group S(P>0.05).However,on postoperative 1 and 3 days,compared with group C,surgery decreased the time traveled by mice in the center of the open field(P<0.05),and this situation did not exist on postoperative 3 days.In fear conditioning test,we found that surgery could significantly decrease the freezing time compared with control group on postoperative 1,3 and 7 days(P<0.05).2.Western blot results showed that NLRP3 protein in the hippocampal tissues of mice in the group S were significantly higher than those in group C on postoperative 1,3 and 7 days,(P<0.05).At these time points,compared with group C,Caspase 1 and ASC in hippocampal tissue of mice in group S were also increased significantly(P < 0.05).Conclusion This study suggested that surgery plus seveflurane anesthesia could lead to postoperative cognitive impairment in mice,which maybe related to the upregulation of NLRP3 inflammasome and its related proteins in the hippocampus of mice,but the specific mechanism needs to be further investigated.Part Ⅱ Honokiol improved postoperative cognitive impairment in mice induced by surgery/seveflurane through activating SIRT3 pathway in the hippocampusObjective To observe the effects of honokiol on the cognitive function of POCD mice and explore the effects of honokiol on the expression of SIRT3,oxidative stress and neuroinflammation in the hippocampal tissues of POCD mice.Methods 1.C57BL/6J mice were randomly split into 7 groups: Control group(group C),Surgery group(group S),surgical anesthesia + honokiol group(group S+HNK),surgical anesthesia + 3-TYP group(group S+3-TYP),Control+honokiol group(group C+HNK),Control+3-TYP group(group C+3-TYP)and surgical anesthesia + honokiol + 3-TYP group(group S+HNK+3-TYP).The mice in the Surgery group received exploratory laparotomy under sevoflurane anesthesia,and the mice in the control group did not undergo surgery.Before the surgery,mice were given honokiol daily for one week at a dose of 10 mg/kg or 3-TYP once every two days for one week at a dose of 50 mg/kg by intraperitoneal injection.The mice in the control group received the same amount(0.5 m L)0.5% dimethyl sulfoxide daily for 7 consecutive days by intraperitoneal injection.On postoperative 1 and 3 days,six mice in each group were underwent behavioral tests,and then were sacrificed to obtain the hippocampal tissues.2.On postoperative 1 and 3 days,the expression of pro-inflammatory cytokines and SIRT3/SOD2 signal related proteins in hippocampal tissue were dectected by Western blott.3.On postoperative 1 day,the apoptosis of neurons in hippocampal CA1 and CA3 was dectected by TUNEL assay.Immunohistochemistry was utilized to find SIRT3 positive cells in hippocampus.The expression of Iba in hippocampus was tested by immunofluorescence.The mitochondrial membrane potential(MMP)was examined by JC-1 kit,according to its protocol.Malondialdehyde(MDA)and mitochondrial radical oxygen species(ROS)levels in the hippocampus were measured with the related kits.Results 1.In open field test,on postoperative 1 and 3 days,there no significant difference in total distance among group C,S,S+HNK and 3-TYP.On 1 day after surgery,compared with the control group,mice in the surgery + sevoflurane group had decreased time in the central area of the open field,while this situation was reversed in the honokiol group(P <0.05).In fear conditioning test,on postoperative 1 and 3 days,mice in surgery+sevoflurane group significantly reduced the freezing time compared with the control group(P <0.05).Honokiol intervention can increase the freezing time compared with the surgery+sevoflurane group(P <0.05).However,3-TYP could reverse the protective effect of honokiol(P <0.05).2.On postoperative 1 and 3 days,in terms of pro-inflammatory,compared with the control group,surgery+sevoflurane can increase the levels of TNF-α,IL-1β and MCP-1 in hippocampal tissue(P<0.05).In addition,surgery+sevoflurane upregulated the expression of Iba-1 in mice’s hippocampal tissue,indicating that surgery+sevoflurane may induced microglia activation in hippocampus(P<0.05).Compared with surgery+sevoflurane group,honokiol intervention can significantly inhibit the levels of pro-inflammatory cytokines,and the expression of Iba-1 in hippocampus(P<0.05);however,3-TYP reversed the effects of honokiol intervention(P<0.05).3.On postoperative 1 and 3 days,compared with the control group,surgery+sevoflurane reduced the expression of SIRT3 protein in the hippocampal tissues of mice,and up-regulated the acetylation level of SOD2(P<0.05).Compared with surgery+sevoflurane group,honokiol intervention could increase SIRT3 expression,and decrease Ac-SOD2 expression in mice’s hippocampus(P<0.05);and 3-TYP can reduce the expression of SIRT3 and increased SOD2 acetylation in hippocampal tissues(P<0.05).According to the results of immunohistochemical detection,compared with the control group,surgery+sevoflurane had fewer SIRT3 positive immune staining cells in the hippocampus(P<0.05);Compared with the surgery+sevoflurane group,honokiol remarkedly increased the number of SIRT3 positive cells in the surgery+honokiol group(P<0.05).However,the 3-TYP treatment inhibited this phenomenon(P<0.05).4.On postoperative 1 day,the number of apoptotic neurons in hippocampal CA1 and CA3 in surgery+sevofluane group was significantly higher than those in control group(P<0.05);honokiol intervention therapy can obviously inhibit apoptosis(P<0.05).However,intraperitoneal injection of 3-TYP can increase neurons apoptosis and eliminate neuroprotective effect of honokiol(P<0.05).5.On postoperative 1 day,compared with the control group,surgery+sevoflurane may raise the levels of ROS and MDA content and decrease the MMP(P<0.05).Compared with surgery+sevoflurane group,honokiol treatment could reverse the above changes(P<0.05).In addition,surgery+sevoflurane can significantly increase the expression of cytochrome C in mice’s hippocampal tissue(P<0.05),and honokiol could reduce the levels of cytochrome C(P<0.05).However,3-TYP treatment could inhibit the antioxidative effect and mitochondrial protective ability of honokiol(P<0.05).Conclusion Honokiol can improve the cognitive decline induced by surgery+ anesthesia exposure in mice.Honokiol could reduce mitochondrial dysfunction and neuroinflammation in the hippocampal tissue of mice through activating SIRT3 signal pathway.Part Ⅲ Honokiol improved postoperative cognitive impairment in mice induced by surgery/seveflurane by mediating mitophagy in the hippocampusObjective To explore the effect of honokiol on mitophagy and NLRP3 inflammasome expression in the hippocampus of mice induced by surgery+sevoflurane.Methods 1.C57BL/6J mice were randomly split into 7 groups: Control group(group C),Surgery group(group S),surgical anesthesia+honokiol group(group S+HNK),surgical anesthesia+3-MA group(group S+3-MA),Control+honokiol group(group C+HNK),Control+3-MA group(group C+3-MA)and surgical anesthesia+ honokiol+3-MA group(group S+HNK+3-MA).The mice in the surgery group received exploratory laparotomy under sevoflurane anesthesia,and the mice in the control group did not undergo surgery.Before the surgery,mice were given honokiol daily for one week at a dose of 10 mg/kg or 3-MA once every two days for one week at a dose of 2 mg/kg by intraperitoneal injection.The mice in the control group received the same amount(0.5 m L)0.5% dimethyl sulfoxide daily for 7 consecutive days by intraperitoneal injection.On postoperative 1,3 and 7 days,six mice in each group were underwent behavioral test,and then were sacrificed to obtain the hippocampal tissues.2.On postoperative 1,3 and 7 days,the expression of NLRP3 related proteins and mitophagy related proteins in hippocampal tissue were dectected by Western blott and ELISA.3.On postoperative 1 day,the apoptosis of neurons in the hippocampal CA1 and DG regions in each group was detected by TUNEL assay.The ultrastructure of mitochondria in hippocampal tissue was observed by transmission electron microscope.NLRP3 positive cells in hippocampus were also detected by immunohistochemistry.Co-expression of LC3,Iba-1 and NEUN in hippocampus were detected by immunofluorescence assay.MDA and ROS levels in the hippocampus were measured with the related kits.Results 1.On postoperative 1,3 and 7 days,for open field tests,there was no significant differences in total distance among these groups.In contextual fear conditioning tests,compared with control group,mice in group S had less freezing time(P<0.05).Honokiol intervention can increase the freezing time and improve the cognitive impairment caused by surgical stress(P <0.05).However,autophagy and mitochondrial autophagy inhibitor 3-MA can reduce the protective effects of honokiol(P<0.05).2.Compared with control group,surgery+sevoflurane can increase the expression of autophagy related proteins LC-3 and Beclin 1 on postoperative 1,3 and 7 days(P<0.05).Compared with surgery+sevoflurane group,surgery+honokiol pretreatment could further enhance the expression of autophagy biomarkers LC-3 and Beclin 1(P <0.05),while autophagy inhibitor 3-MA could significantly reduce the effect of honokiol(P<0.05).3.On postoperative 1,3 and 7 days,Compared with control group,surgery+sevoflurane could increase the expression of mitophagy related protein Parkin and PINK(P<0.05),and honokiol pretreatment could further enhance the expression of these proteins(P<0.05).However,3-MA could decrease the levels of these mitophagy related proteins(P<0.05).Meanwhile,pretreatment with honokiol could significantly reduce the levels of ROS and MDA,which were induced by surgery+sevoflurane(P <0.05),while 3-MA reversed the protective effect of honokiol and induce elevation of ROS and MDA content continuously within 7 days(P<0.05).4.On postoperative 1 day,beclin-1 could be expressed in Iba-1(+)cells and Neu N(+)cells.Compared with the control group,surgery+sevoflurane could promote beclin-1 expression in neurons and microglia cells(P <0.05),while pretreatment with honokiol could further enhance expression of beclin-1 in hippocampal neurons and microglia cells(P <0.05),and 3-MA inhibited the above results(P <0.05).5.On postoperative 1 day,we observed that surgery under sevoflurane could induce mitochondrial structures damage.Compared to healthy cellular organelles in control group,severe mitochondrial damage appeared in the surgery/sevoflurane group.HNK ameliorated the destruction of mitochondria in hippocampus.However,the protective effects were eliminated in the 3-MA intervention group.6.On postoperative 1 day,the number of apoptotic neurons in hippocampal CA1 and DG in surgery+sevofluane group was significantly higher than those in control group(P<0.05);honokiol intervention therapy could obviously inhibit apoptosis(P<0.05).However,intraperitoneal injection of 3-MA could increase neurons apoptosis and eliminate neuroprotective effect of honokiol(P<0.05).Conclusion Honokiol-mediated mitophagy could improve the postoperative cognitive decline of mice induced by surgery/sevoflurane.This neuroprotective effect may involve in inhibiting the activation NLRP3 inflammsome in the hippocampus.