| BackgroundChronic prostatitis/chronic pelvic pain syndrome(CP/CPPS)is characterized by chronic pelvic pain and urethral irritation,accompanied by perineal pain and lower urinary tract irritation such as frequent,urgent and painful urination,as well as sexual dysfunction,long duration,recurrent episodes,difficulty in healing and variable pain sites,which seriously affects the quality of life of patients.However,the pathogenesis of CP/CPPS has not been elucidated and may be related to autoimmunity,chronic congestion and oedema,urinary reflux,oxidative stress and neurological dysfunction.The treatment of CP/CPPS has no specific drugs and is mainly based on symptomatic treatment with alpha-blockers combined with NSAIDs,which is ineffective and takes a long time to treat.Studies have shown that CP/CPPS pain is classified as inflammatory visceral pain and the mechanism may be related to the PKC/NF-κB signalling pathway regulating the expression of P2X3.However,most studies have focused on inflammation,but little has been done on pain symptoms due to CP/CPPS,and the lack of a uniform rat model of CP/CPPS has seriously affected the study of the etiology and pathogenesis of CP/CPPS.Acupuncture points such as Zhong Liao,Hui Yang and San Yin Jiao can effectively relieve pain symptoms,urinary symptoms and improve quality of life in patients with CP/CPPS,and can maintain a better long-term outcome with safety and efficiency and few adverse effects,which has been cited in the 2019 European Association of Urology guidelines as one of the evidences strongly recommending the use of acupuncture therapy for the treatment of CP/CPPS.Previous studies have shown that electroacupuncture in CP rats can promote the normalization of prostate tissue,reduce the expression of inflammatory factors,decrease the production of ROS,and reduce the expression of NF-κB and others,thus providing anti-inflammatory and pain suppression effects.However,the mechanism of acupuncture treatment for CP/CPPS has not been elucidated,but may be related to P2X3 activation and PKC-αphosphorylation,as tissue damage causes Ca2+ inward flow,activates and phosphorylates PKC-α,which in turn sensitizes P2X3 antibodies and promotes NF-κB phosphorylation into the nucleus,and the phosphorylated NF-κB upregulates P2X3 at the transcriptional level,thus causing pain transmission to the central nervous system.Therefore,we propose the scientific hypothesis that "electroacupuncture intervention in CP/CPPS model rats may be related to the regulation of P2X3 expression by PKCα/NF-κB signaling pathway".Objectives and significance1.To construct a CP/CPPS model in rats to assess the stability and temporality of the model in various aspects,so as to make it more similar to clinical type Ⅲa chronic prostatitis.2.To investigate the specific nerve loops that exist between the acupoint commonly used for acupuncture intervention in chronic prostatitis/chronic pelvic pain,the Zhong Xiao acupoint,and the prostate,and to provide an anatomical basis for the neuromodulatory mechanism of acupuncture in the treatment of CP/CPPS.3.To observe the effects of acupuncture in the treatment of CP/CPPS,to explore the possible molecular mechanisms involved,and to provide an experimental basis for the treatment of CP/CPPS with acupuncture.MethodsExperiment Ⅰ:(1)Rats were randomly divided into sham and model groups.The sham group was injected with saline into the ventral lobe of the rat prostate,and the model group was injected with 3%λ carrageenan.(2)The mechanical and thermal pain in the scrotal area of the rats was measured by Von frey fibre wire and thermal pain meter at 72h,10 days,17 days and 31 days after moulding to observe the changes in pain threshold.(3)The prostate index(wet weight/total weight of prostate)was measured after the rats were executed by isoflurane inhalation gas anaesthesia on days 10,17 and 31 after modelling.(4)To assess the degree of inflammation in the prostate tissue of rats,LT-B4 and IFN-γ inflammatory factors were measured by using high-throughput magnetic beads and ultrasonic homogenization technique and Elisa enzyme marker technology.(5)The grey-scale values of PKC-α protein,p-PKC-α protein,NF-κB protein,p-NF-κB,P2X3 protein and SP protein in rat prostate tissue were measured by immunoblotting technique to observe the changes in rat-associated pain proteins.Experiment Ⅱ:(1)Tracer CTB-594 was injected into the mid-shaft point(BL33)with cotton swab pressure for 1min.(2)Tracer CTB-488 2μl was injected into the prostate with gentle cotton swab pressure for 2min,sutured and sterilized on line 4.(3)After 2 d of tracer injection,the tissue of dorsal root ganglia(DRG),spinal cord and sympathetic chain was taken,fixed and sunk in sugar,and confocal microscopy was performed to observe the distribution of tracer CTB-594 and CTB-488 in DRG,spinal cord and sympathetic chain.Experiment Ⅲ:(1)Rats were randomly divided into four groups:sham-operated group,model group,electro-acupuncture group and non-acupuncture group;the shamoperated group was injected with saline into the bilateral ventral lobes of the prostate of rats,the model group,electro-acupuncture group and non-acupuncture group were injected with 3%λ carrageenan,and the electro-acupuncture and sham electroacupuncture interventions were started 72h after surgery.(2)The mechanical and thermal pain in the scrotal area of the rats was measured by using Von frey fibre wire and thermal pain meter for 1 day,4 days,7 days and 14 days after electroacupuncture in each group,and the changes in the pain threshold of the rats were observed.(3)After 14 days of electroacupuncture,the rats were executed by inhalation of isoflurane gas,and the prostate gland was weighed to determine the prostate index(wet weight of prostate/total weight).(4)To assess the degree of inflammation in rat prostate tissue,LT-B4,IFN-γ and inflammatory factors were measured using high-throughput magnetic bead ultrasound homogenization technique and Elisa enzyme marker technology.(5)Paraformaldehyde-fixed tissues,embedded sections and roasted slices were used to determine the expression of PKC-α protein,NF-κB protein,P2X3 protein and SP protein in rat prostate tissue by immunohistochemistry,and to analyze the changes of pain-related proteins in rats.(6)Using immunoblotting technique,PKC-α protein,pPKC-α protein,NF-κB protein,p-NF-κB,P2X3 protein and SP protein grey scale values of rat prostate tissue were measured to study the changes of rat-associated pain proteins.(7)The relative expression of PKC-α,NF-κB,P2X3,SP,P2X7 and CGRP mRNA in the dorsal root ganglion was measured by qPCR technique by taking the DRG of rats bilaterally.ResultsExperiment Ⅰ:(1)The mechanical and thermal pain thresholds of rats in the model group were significantly lower than those in the sham-operated group at 72h,10 days and 17 days after moulding;the mechanical and thermal pain thresholds of rats in the model group were lower than those in the sham-operated group at day 31,but there was no statistical difference in the mechanical pain thresholds between the two groups;this indicated that the mechanical pain symptoms in the model group were maintained for at least 17 days and the thermal pain for at least 31 days.(2)The prostate index of the rats in the model group was higher than that of the sham-operated group on the 10th,17th and 31 st days after moulding,suggesting that the prostate tissue in the model group was more edematous and congested,and the degree of inflammation was more severe.(3)Using the HE technique,the prostate tissue in the model group was disrupted by hyperplasia of the glandular lumen,with a large number of inflammatory cells infiltrating the tissue interstitially,mainly macrophages,and more obvious tissue edema,whereas the prostate structure of the control group was relatively intact,with a small amount of inflammatory cells infiltrating the prostate,without edema and tissue hyperplasia;indicating that the inflammation of the prostate in the model group could be maintained for at least 31 days.(4)The expression levels of LT-B4 and IFN-γ factor in the model group rats were higher than those in the sham-operated group on days 10,17 and 31 after modeling,suggesting that the inflammation of the prostate in the model group rats was more severe and maintained until at least 31 days.(5)The grey-scale values of PKC-α protein,p-PKC-α protein,NF-κB protein,p-NF-κB,P2X3 protein and SP protein in the model group rats were higher than those in the sham-operated group on days 10,17 and 31 after moulding;suggesting that the pain and inflammation in the model group rats may be related to the PKC-α/NF-κB/P2X3 signalling pathway.Experiment Ⅱ:(1)Tracer CTB-594 injected via the Zhongxiao acupoint and tracer CTB-488 injected via the prostate appeared in the same segment of the dorsal root ganglion(DRG);Zhongxiao acupoint tracer-labelled sensory neurons were distributed in the DRG of segments L2-L6,but mainly in segments L4,L5 and L6;prostate tissue CTB-labelled sensory neurons were mainly distributed in the DRG of segments L5 and(2)Both the tracer CTB-594 injected in the middle-shaft and the tracer CTB-488 injected in the prostate appeared in the same sympathetic chain,suggesting that nerve impulses from the prostate and middle-shaft points may be transmitted via sympathetic nerves.(3)Tracer CTB-594 injected into the mid-shaft point labelled sensory neurons appeared in the anterior horn of the spinal cord,whereas no prostate-labelled motor neurons were found in the posterior horn of the spinal cord in rats,mainly because prostate tissue is not under motor nerve control.Experiment Ⅲ:(1)The mechanical and thermal pain thresholds of the rats in the electroacupuncture group were significantly lower than those in the model and nonacupuncture groups at 4,7 and 14 days of electroacupuncture;suggesting that electroacupuncture has the effect of lowering the pain threshold of CP/CPPS rats.(2)After 14 days of electroacupuncture intervention,the prostate index of the rats in the electroacupuncture group was significantly lower than that of the model group and the non-acupuncture group;suggesting that electroacupuncture can reduce the inflammatory degree of prostate tissue.(3)Using HE technique,after 14 days of electroacupuncture intervention,the prostate gland of the rats in the electroacupuncture group was structurally intact,with a lower degree of inflammatory cell infiltration,which was not significantly different from that of the sham-operated group;the interstitial prostate tissue of the model group and the non-acupuncture group had a large amount of inflammatory cell infiltration,some endothelial hyperplasia and more obvious tissue edema;indicating that electroacupuncture could alleviate the degree of inflammation in the CP/CPPS rats.(4)After 14 days of electroacupuncture intervention,the levels of LT-B4 and IFN-γ inflammatory factors in the prostate tissue of the electroacupuncture group were lower than those of the model group and the nonacupuncture group;further supporting the anti-inflammatory effect of electroacupuncture.(5)The expression of PKC-α protein,NF-κB protein,P2X3 protein and SP protein in the prostate tissue of the electroacupuncture group was lower than that of the model group and the non-acupuncture group;suggesting that the mechanism of electroacupuncture intervention in CP/CPPS rats may be related to the regulation of P2X3 expression by PKC-α/NF-κB signaling pathway.(6)The grey values of PKC-αprotein,p-PKC-α protein,NF-κB protein,p-NF-κB,P2X3 protein and SP protein in the prostate tissue of rats in the electroacupuncture group were lower than those in the model and non-acupuncture group;suggesting that the mechanism of electroacupuncture intervention in CP/CPPS rats may be related to the regulation of P2X3 expression by PKC-α/NF-κB signaling pathway.(7)The relative expression of PKC-α,NF-κB,P2X3,SP,P2X7 and CGRP mRNA in the dorsal root ganglion of the electroacupuncture group was lower than that of the model group;further supporting the molecular mechanism of electroacupuncture intervention in CP/CPPS rats from the genetic level.Conclusions1.The CP/CPPS rat model was constructed via prostate injection of carrageenan gum,and its mechanical pain was maintained for at least 17 days,and the heat pain and inflammation level were maintained for at least 3 1 days.2.The existence of nerve loops with similar segments in the DRG and sympathetic chain between the Zhongshao acupoint and the prostate suggests a specific connection between the Zhongshao acupoint and the nervous system,revealing that neuromodulation may be one of the important mechanisms underlying the treatment of CP/CPPS with acupuncture at the Zhongshao acupoint.3.Acupuncture treatment of CP/CPPS was associated with PKC-α/NF-κB signalling pathway regulating P2X3 expression. |
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