| Objective Bupivacaine-induced spinal neurotoxicity can lead to permanent neurological complications,the intrinsic mechanism of which is not known and for which there is no uniform method of prevention or treatment.A rat model of bupivacaine-induced neurotoxicity in the spinal cord was established,to explore the mechanism of NLRP3 inflammasome activation mediated pyroptosis and the ERS-TXNIP-NLRP3 pathway in bupivacaine-induced neurotoxicity.The therapeutic effects of melatonin（MT）on bupivacaine-induced neurotoxicity in the spinal cord was also illustrated.Methods Part I:Experiment in vivo:Sprague-Dawley（SD）male rats（8-10weeks-old,weighing 250-300 g）were randomly divided into 3 groups.Sham operation group（Group C,without intrathecal injection）,saline group（group S,received intrathecal saline）and bupivacaine group（group B,received intrathecal5%bupivacaine）.Each group was analyzed on 12 h,and days 1,3,5,and 7 after the last intrathecal injection（n=8）.The intrathecal injection volume of saline and bupivacaine was 0.12μL/g,administered three times with 90-minute intervals.The percentage maximal possible effect（%MPE）of tail-flick latency（TFL）and Basso-Beattie-Bresnahan（BBB）locomotor scale were used to measure sensation and locomotor function of rats.Hematoxylin-eosin（HE）staining was used to observe histopathological injury and neuronal morphology of the spinal cord in rats.Immunofluorescence was used to evaluate the expression and cellular localization of NLRP3.The protein expression of NLRP3,Caspase-1,cleaved caspase-1,GSDMD,GSDMD-N and IL-1βwas detected by Western Blot.Experiment in vitro:Human neuroblastoma SH-SY5Y cells were treated with different concentrations（0,0.3,0.6,0.9,and 1.2 m M）of bupivacaine.The ultrastructure of SH-SY5Y cells was observed by scanning electron microscope and transmission electron microscope,and the proteine xpression of NLRP3,Cleaved caspase-1,and GSDMD-N was detected by Western Blot.PartⅡ:Experiment 1.Effects of NLRP3 inhibitor MCC950 on the bupivacaine-induced neurotoxicity in spinal cord of rats:Thirty-two SD male rats,8-10 weeks-old,weighing 250-300 g,were randomly divided into 4 groups:Saline group（group SS,received intrathecal saline,followed by intraperitoneal saline once a day for three days）,saline+NLRP3 inhibitor MCC950 group（group SMC,received intrathecal saline,followed by intraperitoneal injection of 5 mg/kg MCC950 once a day for three days）,bupivacaine group（group BS,received intrathecal 5%bupivacaine,followed by intraperitoneal saline once a day for three days）and bupivacaine+NLRP3 inhibitor MCC950 group（group BMC,received intrathecal 5%bupivacaine,followed by intraperitoneal injection of 5 mg/kg MCC950 once a day for three days）;Experiment 2.Effects of TXNIP inhibited adeno-associated virus on the activation of NLRP3 inflammasome in spinal cord of rats:twenty-four male rats,5-6 weeks-old,weighing 100-130g,were randomly transfected with the adeno-associated virus:negative control virus+saline group（group S-sh NC,received intrathecal saline 25 days after intrathecal scramble virus）,negative control virus+bupivacaine group（group B-sh NC,received intrathecal 5%bupivacaine 25 days after intrathecal scramble virus）and TXNIP inhibited adeno-associated virus（AAV-sh TXNIP）+bupivacaine group（group B-sh TXNIP,received intrathecal 5%bupivacaine 25 days after intrathecal AAV-sh TXNIP）.The intrathecal injection volume of the virus was 5μl with a concentration of 1.01×1012 VG/m L.Experiment 3.Effects of ERS inhibitor 4-PBA on the TXNIP-NLRP3 pathway in spinal cord of rats:Thirty-two SD male rats,8-10 weeks-old,weighing 250-300 g,were randomly divided into 4 groups:saline group（group SS,received intrathecal saline,followed by intraperitoneal saline once a day for three days）,saline+ERS inhibitor 4-PBA group（group SP,received intrathecal saline,followed by intraperitoneal injection of 200 mg/kg 4-PBA once a day for three days）and bupivacaine+ERS inhibitor 4-PBA group（group BP,received intrathecal bupivacaine,followed by intraperitoneal injection of 200 mg/kg 4-PBA once a day for three days）;Each group was analyzed on day3.The intrathecal injection volume of saline and bupivacaine was 0.12μl/g,administered three times with 90-minute intervals.HE staining and Nissl staining were used to observe histopathological injury and neuronal morphology of spinal cord in rats.RT-PCR and Western Blot were used to detect the gene and protein expression of TXNIP.The protein expression of NLRP3,cleaved caspase-1,GSDMD-N,IL-1β,PERK,IRE1 and CHOP were detected by Western Blot.Part Ⅲ:SD male rats（8-10 weeks-old,weighing 250-300 g）were randomly divided into 3 groups.Saline group（group SS,received intrathecal saline,followed by intraperitoneal saline once a day）,bupivacaine group（group BS,received intrathecal 5%bupivacaine,followed by intraperitoneal saline once a day）,and bupivacaine+MT group（group BMT,received intrathecal 5%bupivacaine,followed by intraperitoneal injection of 12.5mg/kg MT once a day）.Each group was analyzed on 12 h,and days 1,3,5,and 7 after the last intrathecal injection（n=8）.The intrathecal injection volume of saline and bupivacaine was0.12μL/g,administered three times with 90-minute intervals.The%MPE of TFL and BBB locomotor scale were used to measure the sensation and locomotor function of rats.HE and Nissl staining were used to observe histopathological injury and neuronal morphology of spinal cord in rats.Immunofluorescence was used to evaluate the ratio of neurons with NLRP3.The protein expression of PERK,IRE1,CHOP,TXNIP,NLRP3,cleaved caspase-1,GSDMD-N and IL-1βwas detected by Western Blot.Results Part I:Experiment in vivo:1.Compared with group S,the%MPE of group B was significantly increased（P<0.05）,and BBB score of group B was significantly decreased（P<0.05）,2.Bupivacaine induced injuries for the posterior horn of spinal cord in rats.3.The protein expression of NLRP3,cleaved caspase-1,GSDMD-N and IL-1βin the spinal cord of group B were increased,and highest on day 3（P<0.05）.4.The expression of NLRP3 was mainly in Neu N+neurons of posterior horn in the spinal cord.Experiment in vitro:Bupivacaine induced SH-SY5Y pyroptosis,and,the expression of NLRP3,cleaved caspase-1,and GSDMD-N protein was increased after bupivacaine treatment.PartⅡ:Experiment 1.Effects of NLRP3 inhibitor MCC950 on the bupivacaine-induced neurotoxicity in spinal cord of rats:1.Compared with group BS,the number of survival neurons in group BMC was significantly increased（P<0.05）,and the protein expressions of cleaved caspase-1,GSDMD-N and IL-1βin group BMC were significantly decreased（P<0.05）.Experiment 2.Effects of TXNIP inhibited adeno-associated virus on the activation of NLRP3inflammasome in spinal cord of rats:Compared with group B-sh NC,gene and protein expressions of TXNIP in spinal cord of rats in group B-sh TXNIP were decreased（P<0.05）.AAV-sh TXNIP could inhibit the high expressions of gene and protein of TXNIP after intrathecal bupivacaine.The expression of NLRP3,cleaved caspase-1,GSDMD-N and IL-1βproteins were decreased in group B-sh TXNIP,compared with group B-sh NC,as well as increased the number of survival neurons in the dorsal horn of rats in group B-sh TXNIP.Experiment 3Effects of ERS inhibitor 4-PBA on the TXNIP-NLRP3 pathway in spinal cord of rats:Compared with group BS,the protein expressions of ERS markers PERK,IRE1 and CHOP in the spinal cord of rats in group BP were decreased（P<0.05）.4-PBA could inhibit the high expressions of PERK,IRE1 and CHOP proteins after intrathecal bupivacaine.Compared with group BS,the protein expressions of TXNIP,NLRP3,cleaved caspase-1,GSDMD-N and IL-1βin group BP were decreased（P<0.05）,and the number of survival neurons in the dorsal horn of rats in group BP was increased（P<0.05）.Part Ⅲ:1.Compared with group BS,The%MPE in group BMT decreased from day 3（P<0.05）,and the BBB score increased from day 5（P<0.05）.2.The number of survival neurons in dorsal horn of rats in the BMT group was higher than that in group BS on day 3（P<0.05）.3.The protein expressions of PERK,IRE1,CHOP,TXNIP,NLRP3,Cleaved caspase-1,GSDMD-N and IL-1βin group BMT were lower than those in group BS on day 3（P<0.05）.4.The percentage of NLRP3+neurons in the dorsal horn of rats in the BMT group was significantly decreased than that in group BS on day 3.Conclusions 1.Bupivacaine induces neuronal NLRP3 inflammasome-mediated pyroptosis in the spinal cord of rats.2.Mechanism of ERS-TXNIP-NLRP3 pathway was involved in bupivacaine-induced spinal neurotoxicity.3.MT ameliorates bupivacaine-induced spinal neurotoxicity in rats and suppresses the ERS-TXNIP-NLRP3 pathway in the spinal cord of rats. |
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