The Analgesic Effect Of Multiple-dose Progesterone On Rats With Spinal Nerve Ligation And Its Potential Mechanisms

Background and ObjectiveProgesterone is a progesterone synthesized by the corpus luteum of the ovary,placenta,and adrenal glands.It plays an important role in normal physiology,obstetrics and gynecology related diseases.Progesterone is also a neuroactive steroid.In recent years,it has been reported in the literature that that progesterone can alleviate the nociceptive behaviors induced by chronic constrictive injury(CCI),and progesterone can also inhibit the mechanical and thermal hyperalgesia induced by spinal cord injury,and increase the postoperative thermal pain contraction threshold of sciatic nerve crush injury model rats.However,the mechanisms of progesterone analgesia are still unclear.Research in the past ten years shows that:astrocyte and microglia are activated in neuropathic pain and are critical to the occurrence and development of pain.In addition,protein kinase C γ subtype(PKCγ),as an intracellular signal kinase,is an important mediator of persistent pain behavior and is related to allodynia induced by injury.Activation of PKCγ enhances the phosphorylation of Glutamate N-methyl-Daspartate receptor(NMDAR)in the dorsal horn.The NMDAR subunit NR1 of the dorsal horn is essential for central sensitization induced by nerve injury,and is a key factor in the occurrence and maintenance of chronic pain.The upregulation and phosphorylation of NMDAR accelerate the transmission of primary afferent neurotransmitters to dorsal horn neurons,improving the excitability of sensory neurons.Therefore,this experiment intends to explore the analgesic effect of progesterone on rats with high spinal nerve ligation and its possible relationship with glial cell activation,NMDAR,p-NMDAR and PKCγ expression.Spinal nerve ligation(SNL)was used as the experimental animal,and behavioral method was used to study whether multiple consecutive subcutaneous injection of progesterone has analgesic effect on SNL rats and the duration of the analgesic effect.Cellular immunochemical methods were used to detect the effects of progesterone treatment on glial cells、NMDAR、p-NMDAR and PKCγ in the dorsal horn of the spinal cord of rats with lumbosacral enlargement.Explore the possible relationship between glial cell activation,NMDAR,p-NMDAR and PKC γ expression changes and progesterone analgesia.Exploring the analgesic mechanism of progesterone can provide a new drug target for the treatment of neuropathic pain.MethodsExperiment 1: Preparation of L5 spinal nerve ligation pain modelThirty healthy adult male SD rats were randomly divided into three groups:naive control group,sham operation group and operation group.Before operation,von frey filaments were used to detect the mechanical withdrawal threshold of the three groups of rats,which was measured once a day until the 6th day,and the hyperalgesic and retarded rats were eliminated.Using chloral hydrate anesthetized,the left L5 spinal nerve of the rats in the operation group was ligated,and the rats were recuperated for 2 days after the operation.The mechanical contraction threshold was measured on the 3rd day after the operation until the 21 st day after the operation.The rats in normal control group did not undergo any surgical treatment.The sham-operation group was exposed to L5 spinal nerves without ligation,and the other treatments were the same as those in the operation group.The test result is expressed as 50% PWT(50% paw withdrawal threshold),evaluate the variation of mechanical pain threshold in rats with time.Experiment 2: The effect of multiple consecutive subcutaneous injection of progesterone on the mechanical withdrawal threshold in ratsForty healthy adult male SD rats were randomly divided into four groups:vehicle control group,low-dose progesterone group(8mg/kg),middle-dose group(16mg/kg)and high-dose group(32mg/kg).After L5 spinal nerve ligation,the rats in the control group were injected subcutaneously with 22.5% cyclodextrin solution(3ml/kg)once a day until the 10 th day after the operation.The rats in the experimental groups were injected subcutaneously with 3ml/kg of progesterone(8 mg/kg、16mg/kg and32mg/kg)immediately after the operation,once a day,continued until the 10 th day after the operation.The mechanical withdrawal threshold of experimental rats in each group was measured at 3h post-injection,test every other day until the 31 st day after SNL.Experiment 3: The effect of multiple consecutive subcutaneous injections of progesterone on the expression of microglia,astrocytes,PKCγ,NMDAR1 and p-NMDAR1 in the dorsal horn of the spinal cord of rats with lumbosacral enlargementOn the 5th,17 th and 31 st days after SNL,after measuring the withdrawal threshold,the rats in the naive group,sham operation group,vehicle control group and high-dose(32mg/kg)progesterone group were subjected to cardiac perfusion respectively.The lumbosacral enlargement of the spinal cord was frozen and sectioned.Cellular immunochemical methods were used to observe the changes in the expression of glial cells and PKCγ at the myeloderm,and the co-expression of neuronal cells with NMDAR1 and p-NMDAR1.Results1.The time course of mechanical withdrawal threshold in rats after L5 spinal nerve ligation Over timeAfter L5 spinal nerve ligation,the rats showed foot licking,flicking and lifting the foot,reducing the movement of the hind foot on the surgical side.The mechanical pain threshold results showed that the50%PWT of the hindfoot in the surgical group was significantly reduced on the third day after surgery(P<0.000 vs naive group).Until the 14 th day,the pain threshold was steadily maintained below 6g.From the 15 th day after the operation,the pain threshold showed an upward trend,but until the 21 st day after surgery,the threshold of foot withdrawal on the operation side of SNL rats was still at a very low level(P<0.0001 vs naive group).The mechanical withdrawal threshold did not decrease significantly in the sham operation group(P>0.05 vs naive group).2.Effect of multiple consecutive subcutaneous injection of progesterone on the mechanical withdrawal threshold on SNL ratsMultiple consecutive subcutaneous injection of progesterone significantly increased the mechanical withdrawal threshold of spinal nerve ligation rats.The preoperative mechanical withdrawal thresholds of the vehicle control group,8mg/kg progesterone group,16mg/kg progesterone group and 32mg/kg progesterone group were 15.00±0.00 g,14.61±1.09 g,15.00±0.00 g and 15.00±0.00 g,respectively.The 50%PWT of each group on the 5th day post-operation were 4.59±0.77 g, 4.41±1.20 g,4.89±1.09 g and 6.07±1.09 g respectively.On the 10 th day post-operation, they were 4.71±0.40 g,5.25±1.21 g,8.37±0.93 g respectively and 10.94±1.12 g,respectively.At the 17 d post-operation,they were 5.57±0.51 g, 6.22±0.61 g,9.85±0.71 g and 12.37±0.65 g,respectively.On the 31 th day post-operation,they were 6.49±0.46 g,6.32±0.45 g,7.76±1.47 g and 7.87±0.78g respectively.There was no significant change in the mechanical withdrawal threshold in the low-dose group(8mg/kg)(P>0.05 vs vehicle control group)during the behavior detecting period.From day 7 to day 27 after the operation,the mechanical withdrawal threshold in the middle-dose group of progesterone increased significantly(P <0.05 vs vehicle control group.From day 5 to day 29 after surgery,the mechanical withdrawal threshold in the high-dose group was significantly increased(P <0.01 vs vehicle control group).From day 9 to day 19 after SNL,compared with the middle-dose progesterone group(16mg/kg),the high-dose progesterone group(32mg/kg)significantly increased the mechanical foot withdrawal threshold(P <0.05).Subcutaneous injection of progesterone had no significant effect on the mechanical pain threshold of the contralateral hindfoot.3.Effect of multiple consecutive subcutaneous injection of progesterone on the expression of microglia in the spinal dorsal horn of rats with spinal nerve ligationIn the vehicle control group,the expression of microglia in the dorsal horn of the spinal cord on the surgical side was significantly increased(P <0.05 vs sham operation group),the microglia cell bodies were enlarged,the protrusions became shorter,and changed from branched to amoebic.The mean fluorescence intensity values of spinal dorsal horn microglia in the progesterone group was significantly reduced on the 5th day and the 17 th day after the operation(P <0.001 vs vehicle control group),and there was no significant difference on the 31 st day after the operation.Multiple consecutive subcutaneous injections of progesterone reduced SNL-induced microglia activation.4.Effect of multiple consecutive subcutaneous injection of progesterone on the expression of astrocytes in the spinal dorsal horn of rats after SNLAfter spinal nerve ligation,the expression of astrocytes in the Spinal dorsal horn of rats increased significantly,and the cell bodies were hypertrophy and hyperplasia.The expression level of astrocytes in the normal group and the sham operation group was very low and was at the baseline level.The expression of dorsal horn astrocytes was activated after SNL(P <0.05 vs sham group).Subcutaneous injection of progesterone for 10 consecutive days significantly reduced the expression of astrocytes in the spinal dorsal horn of rats on day 17 after SNL(P <0.05 vs vehicle control group).5.Effect of multiple consecutive subcutaneous injection of progesterone on the expression of PKCγ in the spinal dorsal horn of rats with spinal nerve ligationCompared with the sham operation group,spinal nerve ligation increased the expression of PKCγ in the spinal dorsal horn of rats.Multiple consecutive subcutaneous injection of progesterone significantly reduced the expression of PKCγin the dorsal horn on the 5th day and the 17 th day after SNL(P <0.001 vs vehicle control group).6.Effect of multiple consecutive subcutaneous injection of progesterone on the expression of NMDAR and p-NMDAR on neurons in the spinal dorsal horn of rats with spinal nerve ligationMost of the IR(red)signals of NR1 and p-NR1 colocalized with the green fluorescence signals of Neu N-labeled neurons in the spinal dorsal horn,but not completely.SNL increased the expression of p-NR1 in the spinal dorsal horn(P<0.0001 vs sham operation group).Multiple consecutive subcutaneous injections of progesterone significantly reduced the expression of p-NR1 on the 5th day after SNL(P <0.0001 vs vehicle control group).On the 7th day after the subcutaneous injection of progesterone was stopped,the expression of p-NR1 in the spinal dorsal horn of the progesterone group was still significantly lower than that of the vehicle control group(P <0.01).ConclusionsProgesterone dose-dependently inhibited the mechanical pain behavior induced by spinal nerve ligation in rats,and the analgesic effect could last a long time.The analgesic effect of progesterone may be related to the inhibition of the glial cell activation and the inhibition of the expression of PKCγ and NMDAR1 receptor phosphorylation in the spinal dorsal horn.