The effect of electroacupuncture on the protein expression levels of Cyt-C, Caspase-3 and Caspase-9 in the bladder tissue of rats with neurogenic bladder after suprasacral spinal cord injury

Objective To observe the effect of electroacupuncture on the morphological change of the bladder tissue and the protein expression levels of cytochrome C, Caspase-3 and Caspase-9 in the bladder tissue of rats with neurogenic bladder after suprasacral spinal cord injury, and to preliminarily explore its partial mechanism of action. Methods Forty-eight female SD rats were randomly divided into blank group（group B）, sham operation group（group S）, model group（group M） and electroacupuncture group（group EA） according to random number table method with 12 rats in each group. Neurogenic bladder models after suprasacral spinal cord injury were established by adopting modified spinal cord transection method. Electroacupuncture intervention was conducted at ＂Cìliáo（次髎 BL 32）＂＂Zhōngjí（中极 CV 3）＂ ＂Sānyīnjiāo（三阴交 SP 6）＂ and ＂Dàzhuī（大椎 GV 14）＂ on the 19 th day after modeling. After treatment for 7 consecutive days, the rats were sacrificed, and the bladder tissues were removed rapidly for microscopic observation of morphological change after HE stain and for determination of the protein expression levels of cytochrome C, Caspase-3 and Caspase-9 via Western blot method. Results① Under light microscope after HE stain, injury in varying degrees in muscle fiber arrangement and the shape and size of cells, etc. of bladder detrusor of rats in group M and group EA was found, and the degree of injury of detrusor in group M was severer than that in group EA;② comparison of the protein expression levels of cytochrome C, Pro-Caspase-9 and activated Caspase-9 between group M as well as group EA and group B as well as group S： the protein expression levels of cytochrome C, Pro-Caspase-9 and activated Caspase-9 in bladder tissue of rats in group M and group EA obviously increased（ P〈0.01, P〈0.05）; comparison between group EA and group M： the protein expression levels of cytochrome C, Pro-Caspase-9 and activated Caspase-9 in bladder tissue of rats in group EA obviously reduced（ P〈0.05）;③ comparison of the protein expression level of Caspase-3 between group M as well as group EA and group B as well as group S： the protein expression level of Pro-Caspase-3（35 KD） in group M and group EA reduced, and the protein expression level of activated Caspase-3（17 KD/19 KD） obviously increased（ P〈0.01, P〈0.05）; comparison between group EA andgroup M： the protein expression level of Pro-Caspase-3（35 KD） in group EA obviously increased, and the protein expression level of activated Caspase-3（17 KD/19 KD） obviously reduced（ P〈0.05）. Conclusion suprasacral spinal cord injury can increase the protein expression levels of cytochrome C, activated Caspase-3 and Caspase-9 in bladder tissues of rats, and electroacupuncture at BL 32, CV 3, SP 6 and GV 14 can down-regulate the protein expression levels of cytochrome C, activated Caspase-3 and Caspase-9 in bladder tissues of rats after suprasacral spinal cord injury and protect bladder tissue, which may be one of the mechanisms that electroacupuncture protects the involved bladder detrusor after suprasacral spinal cord injury.

Effects of electroacupuncture on urodynamics,intramedullary apoptosis and bidirectional regulation of neurotrophic factors in neurogenic bladder rats after supersacral spinal cord injury

Objective:To observe the effects of electroacupuncture on urodynamics and intramedullary apoptosis related factor B cell lymphoma gene-2(Bcl-2),Bcl-2 related protein X(Bax)and brain-derived neurotrophic factor(BDNF)in neurogenic bladder rats after supersacral spinal cord injury.Methods:A total of 60 female SD rats,SPF grade,were randomly selected,12 rats in the blank group and 12 rats in the sham operation group,and the remaining 36 rats were made models.Of these 36 rats,24 rats meeting the model standard were randomized into the model group and electroacupuncture group,with 12 rats in each group.The neurogenic bladder models after supracacral spinal cord injury were prepared by the Hassan Shaker spinal cord transection method,via combining the previous model study experience.At the end of spinal shock stage,"Dazhui(大椎GV14)""Zhongji(中极CV 3)""Ciliao(次髎BL 32)",and"Sanyinjiao(三阴交SP 6)"were selected for electroacupuncture.Observation on the histomorphological changes of bladder detrusor and spinal cord in rats was conducted by HE staining.The apoptosis rate of spinal cord cells was measured by TUNEL assay.The maximum bladder volume and compliance were measured by urodynamics test.The protein expressions of Bcl-2,Bax,and BDNF in spinal cord tissues were measured by Western blot assay.The expressions of Bcl-2,Bax,and BDNF mRNA were measured by RT-PCR assay.Results:(1)The results of HE staining showed that compared with the blank group and sham operation group,in the model group the bladder epithelial cell structure was destroyed,the detrusor muscle fibers proliferated,and there were severe bleeding changes,the spinal cord tissue was disordered,the structure was destroyed,the cell space was enlarged,and accompanied by a large number of inflammatory cell infiltration and cystic cavity formation.Compared with the model group,in the electroacupuncture group,the intact bladder epithelial cells increased,the detrusor muscle fiber proliferation decreased,the bleeding changes decreased,the complete structural area of spinal cord tissues increased,the cell space decreased,the levels were clearer,and the cystic cavity decreased.(2)The results of TUNEL assay indicated that the TUNEL positive rate of spinal cord cells in the model group was significantly higher than that in the blank group and sham group(both P<0.05).Compared with the model group,the TUNEL positive rate of spinal cord cells in the electroacupuncture group was significantly reduced(P<0.05).(3)The results of urodynamics test suggested that compared with the blank group and sham operation group,the maximum bladder volume and compliance of the model group were significantly reduced(all P<0.05).Compared with the model group,the maximum bladder volume and compliance were significantly increased in the electroacupuncture group(both P<0.05).(4)The results of Western blot assay indicated that,compared with the blank group and sham operation group,in the model group the protein contents of Bcl-2 and BDNF were significantly reduced(all P<0.05),while the protein content of Bax in the model group was significantly increased(both P<0.05).(5)The results of RT-PCR assay indicated that,compared with blank group and sham operation group,in the model group the intramedullary Bcl-2 and BDNF mRNA were significantly reduced(all P<0.05),and Bax mRNA was significantly increased(both P<0.05).Conclusion:Electroacupuncture at"GV14""CV3""BL32",and"SP6"can promote the repair of bladder detrusor,improve the maximum bladder volume and compliance of bladder,promote morphological repair of bladder detrusor,reduce spinal cord cell apoptosis rate,thus to restore the function of bladder.This is maybe related with the electroacupuncture’s two-way regulatory mechanism of increased expression of protein and gene of Bcl-2 and BDNF and decreased expression of protein and gene of Bax in spinal cord tissues.

Effect of Electroacupuncture on urodynamics, PACAP-38 and PAC1R of spinal cord in rats with neurogenic bladder

Objective： To investigate the effect and mechanism of electroacupuncture（EA） in the intervention of neurogenic bladder（NB） caused by suprasacral spinal cord injury（SCI）.Methods： The total 60 female Sprague-Dawley（SD） rats were divided into blank group, sham operation group by adopting random number table method, with 12 rats in each group. The remaining rats were made into models through operation, and 24 successful model rats were randomly divided into model group and EA group, with 12 rats in each group. Since the 19 th day after modeling, EA was conducted at ＂Cìliào＂（次髎BL 32）, ＂Zhōngjí＂（中极CV 3）,＂Sānyīnjiāo＂（三阴交SP 6） and ＂Dàzhuī＂（大椎GV 14） in EA group, remaining the needle for 20 min every time. The intervention was performed for once a day, and7 consecutive days were needed. The rest of the three groups were only tied up for the same time. Urodynamics test was conducted after the intervention. The contents of PACAP-38 and PAC1 R of the spinal cord of the injury site were detected by Western blot.Results： Compared with the blank group. there were no significant differences in the bladder capacity,leak point pressure, bladder compliance and PACAP-38 and PAC1 R contents in spinal cord in sham operation group; compared with the sham operation group, the maximum bladder capacity and the bladder compliance in the model group were lower（both P〈0.05）, the leak point pressure was higher（P〈0.05）,the contents of PACAP-38 and PAC1 R were lower（both P〈0.05）; compared with the model group, the maximum bladder capacity and the bladder compliance in the EA group were higher（both P〈0.05）, the leak point pressure was lower（P〈0.05）, the contents of PACAP-38 and PAC1 R were higher（both P〈0.05）.Conclusion： EA at BL 32, CV 3, SP 6 and GV 14 can improve the bladder function of NB rats of suprasacral SCI, and its mechanism may be related to the increase expression of PACAP-38 and PAC1 R protein in the injured spinal cord tissue.

Effect of electroacupuncture at“ Zúsānlǐ”(足三里 ST 36) and other lower he-sea points on the expression of TNF-α and NF-κB of acute gastric mucosal lesion rats

Objective To contrastively observe the effect of electroacupuncture at lower he-sea points of stomach, large intestine, small intestine and gallbladder of acute gastric mucosal lesion（AGML） model rats on the expression of serum high mobility group protein B 1（HMGB 1）, tumor necrosis factor-α（TNF-α） and nuclear factor-κB（NF-κB） of gastric tissue, and to explore whether the lower he-sea points of stomach have relative specificity in treatment of stomach diseases. Methods Seventy healthy SD rats were randomly divided into seven groups, including control group, model group, ＂Zúsānlǐ＂（足三里 ST 36） group, ＂Shàngjùxū＂（上巨虚 ST 37） group, ＂Xiàjùxū＂（下巨虚 ST 39） group, ＂Yánglíngquán＂（阳陵泉 GB 34） group and ＂Chéngjīn＂（承筋 BL 56） group, with five male rats and five female rats in each group. Acute gastric mucosal lesion model was established by applying WRS method. After successful modeling and treatment for ten days, sampling was conducted, gastric mucosal lesion was observed visually and scored, the levels of serum HMGB 1 and gastric tissue TNF-α were detected by ELISA method, and the expression of NF-κB was detected by immunohistochemical method. Results①Compared with the model group, the levels of Ulcers and inflammation（UI） in gastric mucosal and serum HMGB1, and the expression of gastric tissue TNF-α and NF-κB in the four lower he-sea points groups reduced（P〈0.01 or P〈0.05）;② compared with ST 36 group, the levels of gastric mucosal UI and serum HMGB1, and the expression of gastric mucosal TNF-α and NF-κB in other treatment groups increased significantly, and the difference was statistically significant（P〈0.01 or P〈0.05）. Conclusion①Electroacupuncture at relevant lower he-sea points can inhibit the body＇s immune response, relieve inflammatory reaction and reduce mucosal lesion by regulating HMGB 1, TNF-α and NF-κB, so as to realize the intervention and treatment on AGML rats;② the total intervention effect in ST 36 group is superior to that in other groups, indicating partially that there is a relative specificity between ST 36 and stomach.

A study on the differences of effect on mast cells and serum 5-HT in the acupoint area of "CV 4" of rats by acupuncture stimulus and thermal stimulus

Objective: To observe the influence of acupuncture stimulus and thermal stimulus on the expression of mast cells(MCs) and 5-hydroxytryptamine(5-HT) in local acupoint area of Guanyuan(关元 CV 4) and serum 5-HT in rats.Methods: Thirty-nine male C57 BL/6 rats were randomly divided into blank control group, acupuncture stimulus group and thermal stimulus group, with 13 rats in each group. The rats were stimulated by manual acupuncture at CV 4 for 5 min in acupuncture stimulus group, while those in thermal stimulus group were stimulated by adopting a thermal moxibustion apparatus on CV 4 for 30 min. The expression of MC and 5-HT in the skin in the acupoint area of CV 4 before and after acupuncture stimulus and thermal stimulus was observed and analyzed via adopting toluidine blue staining method and immunofluorescence histochemical method(5 rats were selected from each group), and the 5-HT content in serum before and after stimulus was determined through enzyme-linked immunosorbent assay(ELISA)(8 rats were selected from each group). The influence of acupuncture stimulus and thermal stimulus on the contents of MC and 5-HT in the skin and serum 5-HT in rats was analyzed and compared.Results: ① After acupuncture stimulus or thermal stimulus on CV 4 of C57 BL/6 rats, the number of MC in the acupoint area significantly increased when compared with that in blank group(the acupuncture stimulus group 12.40 ± 2.07 vs. the blank group 3.00 ±5.96;thermal stimulus group 26.20 ± 10.85 vs. the blank group 12.40 ± 2.07, both P< 0.05), and MC aggregation and degranulation were observed(the acupuncture stimulus group 17.80 ±4.55 vs. the blank group 8.00 ±3.16;the thermal stimulus group24.00±9.05 vs. the blank group 8.00±3.16. P<0.05, P< 0.01).② After acupuncture stimulus or thermal stimulus, 5-HT was released by MCs in the acupoint area, which aggregated around the blood vessels,and the number of 5-HT in the acupoint area significantly increased when compared with that in blank group(the blank group 3.00 ±1.28 vs. the acupuncture stimulus group 10.02 ±3.21;the blank group3.00 ±1.28 vs. the thermal stimulus group 14.00 ±3.94, both P< 0.01).③ Compared with blank group,both acupuncture stimulus and thermal stimulus could reduce the 5-HT content in serum(the blank group 0.72 ±0.2372 vs. acupuncture stimulus group 0.43 ±0.21: the blank group 0.72 ±0.24 vs. thermal stimulus group 0.32 ±0.18, both P<0.01), and the effect in thermal stimulus group was slightly superior to that in acupuncture stimulus group(P<0.05).Conclusion: Both acupuncture stimulus and thermal stimulus can cause the aggregation and degranulation of MCs and high expression of 5-HT in the acupoint area. The effect of thermal stimulus was superior to that of acupuncture stimulus in degranulation.

Effect of moxibustion on survival status and nutritional metabolic factor in tumor-bearing rats with gastric cancer

Objective:To observe the effect of moxibustion on the survival status and nutritional metabolic factors of tumor-bearing rats with gastric cancer and explore the inhibitory effect of moxibustion on the tumorbearing rats.Methods:A total of 40 SD rats were randomly divided into a blank group,a sham-operated group,a model group and a moxibustion group,10 rats in each group.Gastric cancer models were established in the model group and the moxibustion group.The rats of all the groups underwent the same constraining procedure for 20 min every day.Additionally,moxibustion was applied at acupoints for 20 min in the rats of the moxibustion group.There were two groups of acupoints.One group included"Zusanli(足三里ST 36)""Zhongwan(中脘CV12)"and"Guanyuan(关元CV4)".The other group included bilateral"Pishu(脾俞BL 20)"and"Weishu(胃俞BL 21)".Moxibustion was applied for 20 min every day at the acupoints of the two groups alternatively and lasted for 14 days.The survival status of rats was observed and the score of survival status and body weight of rats were recorded every day.At the end of intervention,the orbital blood was collected and the rats were sacrificed for sample collection.The viscera-free body weight was recorded.Using biochemical analyzer,the blood glucose(GLU),albumin(ALB),total protein(TP)and triglyceride(TG)in serum were detected.Results:(1)Regarding the score of the survival status,after modeling,the scores in the model group and the moxibustion group were all higher than those of the sham-operation group(both P<0.05).After intervention,the score in the moxibustion group was lower than that of the model group(P<0.05).(2)After modeling,the body weight in the moxibustion group and the model group was lower than those of the blank group,indicating a statistical significance(both P<0.05).The body weight in the moxibustion group was higher than that of the model group after intervention,indicating the statistical significance(P<0.05).(3)Regarding the viscera-free body weight,it was lower in the model group compared with the sham-operation group,indicating the statistical significance(P<0.05).Compared with the model group,the viscera-free body weight was higher in the moxibustion group,indicating the statistical significance(P<0.04).(4)Regarding the nutritional metabolic factors,compared with the sham-operation group,the levels of GLU and ALB in the model group were lower and TP was higher,presenting the statistical significance(all P<0.05).Compared with the model group,the levels of GLU and ALB were higher in the moxibustion group and TP was lower,presenting the statistical significance(all P<0.05).Conclusion:Moxibustion improves the survival status of tumor-bearing rats with gastric cancer,increases the body weight,the viscera-free body weight and the levels of GLU and ALB in serum and reduces TP of the tumor-bearing rats with gastric cancer and inhibits the growth of gastric cancer.

Analysis of the differences in brain function response after acupuncture and moxibustion at Zusanli(足三里ST36)in the patients with functional dyspepsia based on fractional amplitude of low frequency fluctuation of rest functional magnetic resonance imaging

Objective:To explore the differences in the central response after acupuncture and moxibusbution at Zusanli(足三里ST36) in treatment of functional dyspepsia(FD) based on the analysis of fractional amplitude of low frequency fluctuation(fALFF) of resting-state functional magnetic resonance imaging(rfMRI).Methods:A total of 60 patients with FD were randomized into an acupuncture group and a moxibustion group,30 cases in each one.In the acupuncture group,acupuncture was applied to bilateral ST36.In the moxibustion group,moxibustion was exerted at bilateral ST36.The treatment was given once a day,5 times a week,totally for 4 weeks in each group,including 20 treatments with acupuncture or moxibustion.Separately,before and at the end of treatment,rfMRI scanning was conducted in two groups.Using data processing assistant for resting-state fMRI(DPARSF) software and MATLAB data platform,rfMRI data were collected for preprocessing and fALFF analysis.Results:Compared with the data before treatment,after treated with acupuncture at ST36 in FD patients,fALFF value was reduced in the right superior frontal gyrus,left superior frontal gyrus,left inferior frontal gyrus,right cuneus,left precuneus,right middle occipital gyrus,left middle occipital gyrus,etc.,and it was increased in the left parahippocampus,right parahippocampus,left cerebellum,etc(all P <0.01).After treated with moxibustion,the remarkable increase of fALFF was not discovered in brain areas,but fALFF decreased significantly in the left superior parietal gyrus(P <0.01).Compared with moxibustion at ST36,after acupuncture at ST36 in FD patients,the increase of fALFF appeared in the right putamen and the decrease of it occurred in the middle occipital gyrus,indicating the statistical significance(both P <0.05).Conclusion:The differences in central function responses are induced in treamtent with acupuncture and moxibustion,which is probably related to the resting-state default network associated with targeted regulation and ventral attention of brain areas in functional dyspepsia.

Periarthritis of shoulder treated with Fu’s subcutaneous needling and electroacupuncture:A controlled trail

Objective:To observe and compare the therapeutic effect on shoulder periarthritis between Fu’s subcutaneous needling(FSN)and electroacupuncture(EA).Methods:A total of 68 patients with periarthritis of shoulder were divided into a FSN group and an EA group according to the random number table,34 cases in each one.In the FSN group,FSN was adopted,once every two days,totally for 10 times.In the EA group,EA was used once daily,consecutively for 20 times.Shoulder pain score and shoulder function score(Constant-Murley score)before and after treatment were compared between the two groups.The clinical therapeutic effect was analyzed and evaluated statistically.Results:After 20-day treatment,shoulder pain score was reduced obviously as compared with that before treatment in the patients of either group(both P<0.01).After treatment,shoulder pain score in the FSN group was lower significantly than that in the EA group(P<0.01).In 20 days of treatment,CMS was improved obviously as compared with that before treatment in the patients of both groups(both P<0.01)and CMS in the FSN group was higher significantly than that in the EA group after treatment(P<0.01).The effective rate was 94.11%in the FSN group and was 91.17%in the EA group.The remarkably effective rate was 73.52%in the FSN group,significantly higher than 20.58%in the EA group(P<0.01).Conclusion:Both FSN and EA are effective in treatment of periarthritis of shoulder,but FSN obtains a much higher remarkably effective rate in treatment of periarthritis of shoulder and its effects for relieving shoulder pain and improving shoulder function are superior to EA.Because of its effective treatment and simple operation,FSN deserves to be promoted in clinical practice for periarthritis of shoulder.

Hypercholesterolemia treated with medicinal pad-separated moxibustion:A randomized clinical trial

Objective:To assess the effect of medicinal pad-separated moxibustion for reducing low-density lipoprotein cholesterol(LDL-C)in the patients with hypercholesterolemia.Design:Clinical randomized controlled trial with blinding for outcome assessors and statisticians.Setting:Department of Acupuncture-Moxibustion and Tuina,Department of Physical examination of the First Affiliated Hospital of Hunan University of Chinese Medicine and three community health centers adjacent to the Hospital and University,from July 2015 to December 2017.Patients:60 patients with hypercholesterolemia(elevated LDL-C).Interventions:The therapeutic lifestyle change(TLC)was provided in both groups.In the experiment group,medicinal pad-separated moxibustion was applied using two groups of acupoints(Group No.1:Jùquē[巨阙CV14],Tiānshū[天枢ST25]and Fēnglóng[丰隆ST40]).(Group No.2:Xīnshū[心俞BL15],Gānshū[肝俞BL18]and Píshū[脾俞BL20])alternatively for 3-5 times a week.In the control group,Simvastatin tablets were administered orally by the patients in accordance with instructions in the medication guides(10 mg/d).Main outcome measures:Change of LDL-C after 12 weeks of treatment compared with the baseline.Results:After 12 weeks of treatment,LDL-C was reduced in both the medicinal pad-separated moxibustion group and the simvastatin group compared with that at baseline(both P<0.05).The difference on change of LDL-C was not significant between groups(P>0.05).At week 4 of follow-up,LDL-C was reduced compared with that at baseline in the medicinal pad-moxibustion group(P<0.05),and the difference on change of LDL-C was significant between groups(P<0.05).At week 12 of follow-up,the difference on change of LDL-C was not significant when compared with the baseline in each group(both P>0.05)and between groups(P>0.05).In comparison with values before treatment,the values of triglyceride(TC)and triacylglycerol(TG)were reduced in both groups(all P<0.05),while value of highdensity lipoprotein cholesterol(HDL-C)was increased in the medicinal pad-separated moxibustion group after 12 weeks of treatment(P<0.05).The change of TG and HDL-C values were significantly different between groups(both P<0.05).At week 4 of follow-up,values of TC and TG were lower(both P<0.05)and HDL-C was higher(P<0.05)when compared with that at baseline in the medicinal pad-separated moxibustion group;and value of TC was lower and HDL-C higher in the medicinal pad-separated moxibustion group compared with that in the simvastatin group(both P<0.05).At week 12 of follow-up,compared with that at baseline,all blood lipid outcomes were not significantly different either within(P>0.05)or between groups(P>0.05).Conclusion:Medicinal pad-separated moxibustion could reduce LDL-C and increase HDL-C in patients with hypercholesterolemia.However,these results need to be further verified by study with large sample size.

Strategies of the acupuncture treatment for delayed-onset muscle soreness based on pathophysiology

Based on the pathophysiological analysis of delayed-onset muscle soreness,the key points of its treatment was summarized in this paper:promoting the repair of injury,improving the metabolic state,and effectively relieving pain.Its key objectives and order of treatment are made clear:first to promote repair,second to improve metabolism,and then to relieve pain.And through the analysis of the key points of the treatment,the law of acupuncture and moxibustion treatment is put forward as follows:puncture those acupoints with the overall regulation function to adjust the secretion of endogenous analgesic substances in the body,at the same time,the special respiratory movement is adopted to improve the metabolic state,and the proper exercises are combined to promote the tissue repair.