| Background:Pain has brought great suffering to modern humans,and seriously affected people's quality of life.Chronic pain is a common concomitant symptom of many diseases.Worldwide,there are far more patients with chronic pain than those with diabetes,heart disease and cancer.However,due to the complex mechanism of chronic pain,including inflammatory factors,nerve or tissue damage factors,etc.,clinically there is still no effective treatment method and drug with few side effects for treating chronic pain.According to the recommendation of the World Health Organization(WHO),opioids are the first choice for the treatment of moderate to severe chronic pain,such as cancer pain.Opioids,such as morphine and fentanyl,are the most commonly used and most effective type of analgesics in clinical practice.However,opioids can cause many side effects such as constipation,itching,and respiratory depression when used.At the same time,long-term and repeated use of opioids will inevitably lead to drug resistance,which gradually reduces or even disappears the analgesic effect of the drug,and produces hyperalgesia caused by opioids,and clinical treatment in order to achieve the original The pain effect has to gradually increase the dose of the drug,which will undoubtedly increase the side effects and adverse reactions of the drug,and even lead to the development of drug addiction.So,how can we maximize the analgesic effect of opioids while avoiding adverse reactions such as drug tolerance and addiction?This is a hot issue that researchers and clinicians worldwide are concerned about.However,due to the unclear mechanism leading to opioid tolerance and addiction,there is still no method that can effectively prevent the development of this type of drug tolerance addiction,which greatly limits the clinical use of opioids,thereby reducing The quality of life of patients with acute and chronic pain.To clarify the mechanism of opioid tolerance and addiction,to explore effective prevention and treatment methods,and to improve the quality of life of patients with chronic pain,especially moderate to severe chronic pain,are very urgent scientific research and medical tasks.Unfortunately,due to the complex mechanism of morphine tolerance,it is not clear so far.Therefore,there is currently no effective way to prevent or treat it.This study is divided into three parts,mainly through behavioral testing.Molecular biological detection and other means,to preliminary explore the Shh signaling pathway in the development and development of morphine tolerance and its regularity,and the impact on morphine tolerance.The aim is to provide a new idea for exploring the mechanism of morphine tolerance and a new target for the treatment of morphine tolerance.Part 1 The expression and distribution of Shh signaling pathway in morphine toleranceObjective:To establish a model of morphine tolerance by using molecular biology and morphological techniques,and to investigate the temporal and spatial variation of Hedgehog signaling pathway expression in the spinal cord of mice during morphine tolerance,as well as the behavioral effects of such expression and morphine tolerance.Methods:Adult male C57 mice with body weight(22-24 g)and aged 8-10 weeks were randomly divided into two groups:Sham group and morphine tolerance group(Mor group).The Mor group was further divided into several subgroups according to different time points(8 mice in each group).Referred to the literature,the chronic morphine tolerance model was established by repeated subcutaneous injection of morphine 10 mg/kg once a day for 7 consecutive days.The Sham group was injected with normal saline of equal volume at the same time point.Two hours after each injection,the maximum analgesic effect of morphine(MPE%)and the change of threshold of heat-shrinkable foot were measured by hot plate method and thermal stimulation method per day for 7 consecutive days,Further,n the determination of the behavioral changes,animals in subgroup at different time points(1,3,5,7 day),were killed,and quickly removed the spines of L4-L6 segment and bilateral DRG,to detect SHH signaling pathways important protein molecules(intracytoplasmic SHH,Ptch1,Smo,and Gli1 in the cell nucleus expression changes using western blot technique.At the same time,the cytological localization of increased Shh in the spinal cord was further clarified by using the immunofluorescence double-labeling technique.Since the Shh protein molecule is a secretory protein,which can only function if it is synthesized and secreted,we used ELISA technology to detect the changes in the concentration of Shh in spinal cord tissue and DRG to reflect the secretion of Shh.Results:After repeated injection of morphine,the experimental animals gradually showed morphine tolerance.Mainly with repeated injection of morphine,morphine analgesia effect(MPE%)largest gradually reduced,from the third day after morphine injection appeared significantly decreased(P<0.05),to continuous morphine injection after 7 days,not only the MPE%decreased significantly,and mice also showed obvious lower hot shrinkage threshold(P<0.05).Subsequently,western blot analysis showed that the expression of Shh and its receptor Ptch1 and Smo in spinal cord and DRG tissues increased in a time-dependent manner with the repeated injection of morphine,and the change trend was significantly consistent with the decrease of MPE%and heat-shrinkable foot threshold.In addition,with the repeated injection of morphine,the expression of Glil in the nucleus also increased in a time-dependent manner,which suggests that the activation of Shh signaling pathway was gradually enhanced.Meanwhile,ELISA results showed that the concentration of Shh protein molecules in spinal cord tissues and DRG tissues significantly increased after repeated injection of morphine,indicating that a large amount of Shh protein was secreted.Furthermore,through immunofluorescence,we found that increased Shh was mainly expressed in the middle and small neurons related to the transmission of harmful information in DRG tissue,and similarly,increased Shh was mainly expressed in the i-ii and v-vi plates receiving the transmission of harmful information in the dorsal horn of the spinal cord.Through fluorescence double labeling,it was found that Shh was mainly expressed in neurons and astrocytes,but hardly expressed in microglia.Conclusion:Repeated injection of morphine can induce increased expression and activation of Shh signaling pathway in spinal dorsal horn and DRG.Increased Shh is mainly distributed in pathways related to the transmission of harmful information.Cytologically,increased Shh is mainly expressed in neurons and astrocytes,but almost not in microglia.Part 2 The effect of Shh signaling pathway interference on morphine tolerance behavior and neurochemistryObjective:To investigate the effect of inhibition of Shh signaling pathway on the development of morphine tolerance and neurochemical changes in spinal cord.Methods:Adult male C57mice,weighted(22 to 24 g)and aged 8 to 10 weeks,were randomly divided into 4 groups:group Mor(repeated subcutaneous injection of morphine,once a day,seven days),Sham group(such as subcutaneous injection volume of physiological saline),CLP+Mor group(model group in morphine tolerance by intraperitoneal injection of different time points of cyclopamine,once a day,three days in a row,as shown in the body),DMSO+Mor group(model group in morphine tolerance of different point in time,injection volume and the CLP solvent of DMSO),10 mice in each group.In order to further confirm the role of Shh signaling pathway,we also used Shh siRAN,a small interfering RNA of Shh signaling pathway,and control siRNA(con-sirna)as control.Intrathecal injection of Shh siRNA(1 g/10ul,once a day for 3 consecutive days)was given 3 days prior to morphine injection.Behavioral and molecular biology techniques were used to detect the effects of Shh signal pathway interference on behavioral changes of morphine tolerance and neurochemical changes in the spinal cord.Results:Early intraperitoneal injection of CLP(10 mg/kg,once a day,for 3 consecutive days)at the first,second and third day of repeated morphine injection significantly delayed the development of morphine tolerance and alleviated the degree of morphine tolerance.The onset of MPE%decline was significantly delayed(from day 3 to day 5-6),and the magnitude of decline was significantly reduced(P<0.05).At the same time,the time of the decrease of the threshold value of heat shrinkable foot in mice was also significantly delayed,and the range of decrease was also significantly reduced(P<0.05).However,CLP administration at day 5,6,7(late)of repeated morphine injection had no significant effect on the resulting MPE%decline and thermal foot threshold decline(P>.05).To further verify this result,we used the biological interference agent Shh siRNA.Similar to drug inhibition,after Shh siRNA injection in advance,morphine tolerance induced by repeated morphine injection was significantly delayed and alleviated,and the rate and magnitude of MPE%decline were significantly reduced(P<0.05).Meanwhile,molecular biological test results showed that the expression of c-fos protein and CGRP protein in spinal dorsal horn increased significantly after repeated injection of morphine,and the inhibition of Shh signaling pathway by cyclopamine(day 1,2 and 3)in advance could significantly inhibit the up-regulation of c-fos and CGRP expression in spinal dorsal horn caused by repeated injection of morphine.Conclusion:Inhibition of Shh classical pathway can effectively inhibit morphine tolerance and the generation of morphine induced hyperalgesia,but has no significant inhibitory effect on the generation of morphine tolerance.Blocking the Shh signaling pathway inhibited neurochemical changes in the spinal cord caused by morphine tolerance.Part 3 Shh signaling pathway participates in the regulation ofmorphine tolerance by upregulating brain-derived neurotrophic factor(BDNF)Objective:To explore the possible mechanism of Shh signaling pathway regulating morphine tolerance by using Inhibitors and agonists of the Shh signaling pathway and inhibitors and neutralizing antibodies of BDNF.Methods:Referred to the literature,firstly,Shh drug inhibitor CLP and small interfering RNA Shh siRNA were injected into the morphine tolerance model respectively(the administration method was the same as the second part),and the expression changes of BDNF protein in spinal cord tissues were detected using molecular biology techniques(n=6).Then,in normal mice,exogenous Shh signaling agonist SAG(5mg/kg,i.p.)was given to detect the effect of activation of Shh signaling pathway on BDNF expression in the spinal cord,as well as the effect on thermal foot threshold in mice,using molecular biology techniques and behavioral techniques.Finally,in the morphine tolerance model,the inhibitor K252(80 g/kg,i.t.)of BDNF or the antibody anti-bdnf(2 g/kg,i.t.)were injected into BDNF or BDNF to neutralizing the antibody anti-bdnf(2 g/kg,i.t.)Results:Western blot results showed that the expression of BDNF protein in spinal cord tissues increased in a time-dependent manner after repeated injection of morphine.It was significantly increased from day 3 after repeated morphine injection(P<0.05),and peaked on day 5-7.The inhibition of Shh signaling pathway by CLP or Shh siRNA can effectively inhibit the up-regulation of BDNF expression in spinal cord tissues caused by repeated morphine injection.In normal animals,exogenous injection of the Shh signaling agonist SAG rapidly(1 hour after injection)induces up-regulation of BDNF expression in spinal cord tissues,which can be inhibited by CLP.In addition,injection of SAG into normal animals significantly reduced the threshold of heat-withdrawal foot in experimental animals(P<0.05),while the effect of SAG could be reversed by K252 or anti-bdnf.In addition,early administration of BDNF inhibitor K252 or anti-bdnf(once daily for 3 consecutive days)effectively delayed morphine tolerance and MIH production,and significantly reduced MPE%and heat-withdrawal foot threshold.The co-expression of BDNF in Shh protein and Glil protein was confirmed by immunofluorescence double labeling in the dorsal horn of spinal cord.Conclusion:Repeated injection of morphine can up-regulate the expression of BDNF in spinal cord tissue,and inhibition of BDNF can effectively delay and alleviate the generation of morphine tolerance,BDNF may be a target protein downstream of the Shh signaling pathway,which may regulate morphine tolerance by up-regulating BDNF in spinal cord tissue. |
PDF Full Text Request