| Objective: To observe the effects of intrathecal injection of Endoglin recombinant protein on pain behavior in spared nerve injury(SNI)rats,and investigate whether it affects neuropathic pain by regulating the microglia activation and expression of pro-inflammatory factors in spinal cord dorsal horn through phosphorylation of TGF-β1/Smad2 signaling pathway.Methods: 1: Twelve sprague-Dawley(SD)male rats(200-220 g,6-8weeks of age)were randomly divided into two groups,each with six rats,namely Sham group(Sham)and model group(SNI).In the SNI group,the right tibial nerve and common peroneal nerve were ligated and cut off with 4-0 silk thread to establish the SNI model,while in the Sham group,the nerve was only exposed without ligation.The rats were killed at 14 days after modeling,and the expression of Endoglin in spinal cord of the two groups was detected by Western blot after the paw withdrawal threshold(PWT)and thermal withdrawal latency(TWL)detection.2: A total of 150 male SD rats were randomly divided into three groups,each with fifty rats,including Sham operation + intrathecal injection of normal saline group(Sham+NS),model + intrathecal injection of normal saline group(SNI+NS)and model + intrathecal injection of Endoglin recombinant protein group(SNI+ENG).The rats in SNI+NS group and SNI+ENG group were performed with Spared nerve injury(SNI)model;The rats in Sham+NS group only exposed the nerve without injury.The SNI+ENG group was given 20μl of Endoglin recombinant protein(2μg/ml)every other day from the first day to the 14 th day after model establishment,and the other groups were given the same amount of normal saline at the same time point.Behavioral tests were performed on 1 day before SNI(T0),3 days after SNI surgery(T1),7 days after SNI surgery(T2),and 14 days after SNI surgery(T3),which including PWT and TWL.The rats were killed at T1,T2 and T3 point,respectively,and immunofluorescence staining was used to observe the activation of microglia in L4-5 spinal dorsal horn during T1-T3.The protein expressions of TGF-β1,Smad2 and p-Smad2 in L4-5 spinal dorsal horn at T3 were detected by Western blot.The levels of TNF-α,IL-1β and IL-6 in L4-5spinal cord were detected by ELISA during T1-T3.Results: 1: On the 14 th day after SNI,the expression of Endoglin in the spinal cord of the SNI group was significantly lower than that of the Sham group(P<0.05).2: The SNI rats showed pain sensitization from T1,and the PWT and TWL of the SNI+NS group and SNI+ENG group were significantly lower than those of Sham+NS group at T1-T3(P<0.05);However,intrathermal injection of Endoglin recombinant protein significantly improved the pain intensity of SNI rats.Compared with SNI+NS group,PWT and TWL of SNI+ENG group were significantly increased at T1-T3(P<0.05).In addition,microglia in the L4-5 spinal cord of SNI+NS were significantly activated at T1-T3,and the activation was most obvious at T2.Compared with the SNI+NS group,the activation of microglia inhibited in SNI+ENG group by the Endoglin recombinant protein.At T3,the expressions of TGF-β1 and p-Smad2 protein in SNI+NS group were significantly decreased compared with Sham+NS group(P<0.05).Compared with the SNI+NS group,the expressions of TGF-β1 and p-Smad2 protein in SNI+ENG group were significantly increased(P<0.05).Compared with Sham+NS group,the expression of Smad2 protein in SNI+NS group were increased(P<0.05),while there was no statistical significance in the expression of Smad2 between SNI+ENG group and SNI+NS group(P>0.05).Moreover,the release of TNF-α,IL-1β and IL-6 in l4-5 spinal cord of rats after SNI was significantly increased.TNF-α,IL-1β and IL-6 in the spinal cord of rats in the SNI+NS and SNI+ENG groups were significantly up-regulated at T1-T3 compared with that in the Sham+NS group(P<0.05),but the up-regulated of TNF-α,IL-1β and IL-6 was significantly inhibited in SNI+ENG group(P<0.05).Conclusion: 1.Endoglin expression was decreased in spinal cord of NPP animal model.2.SNI surgery could cause hyperalgesia in rats,and intrathecal injection of Endoglin recombinant protein could relieve hyperalgesia in SNI rats.3.SNI surgery could induce microglia activation,increase the release of pro-inflammatory cytokines,and promote neuroinflammation,while intrathecal injection of Endoglin recombinant protein could inhibit microglia activation and reduce the secretion of pro-inflammatory factors.4.TGF-β1/Smad2 signal was inhibited in the spinal cord of rats after SNI surgery,but was activated after intrathecal injection of Endoglin recombinant protein,suggesting that TGF-β1/Smad2 signal may be involved in the inhibition of microglia activation and thus affect the development of neuroinflammation and neuropathic pain. |
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