Mechanisms of acupuncture for primary dysmenorrhea based on PI3K/Akt/mTOR signaling pathway in rats. / 基于PI3K/Akt/mTORä¿¡å·é€šè·¯æŽ¢è®¨ç”µé’ˆæ²»ç–—åŽŸå‘æ€§ç—›ç»å¤§é¼ çš„æœºåˆ¶.

OBJECTIVES: To observe the effect of electroacupuncture(EA) on phosphatidylinositol-3-kinases(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR) signaling pathway of uterus tissue in rats with primary dysmenorrhea(PDM), so as to investigate its mechanisms underlying improvement of PDM. METHODS: Thirty healthy non-pregnant female SD rats were randomly divided into blank, model and EA groups, with 10 rats in each group. The PDM model was established by subcutaneous injection of estradiol diphenhydrate combined with intraperitoneal injection of oxytocin. For rats of the EA group, EA(50 Hz, a tolerable current intensity) was applied to "Guanyuan"(CV4) and bilateral "Sanyinjiao"(SP6) for 20 min, once a day for 10 consecutive days. The number of writhing, wri-thing score, and writhing latency were observed. The uterine histopathological changes were observed by H.E. staining, and the ultrastructural changes of uterine tissue cells in each group were observed by transmission electron microscopy. The contents of prostaglandin E2(PGE2), prostaglandin F2α(PGF2α) and ratios of PGF2α/PGE2 in the serum and uterine tissue were detected by ELISA. The relative expression levels of PI3K, Akt and mTOR and their phosphorylation proteins in the uterine tissue were detected by Western blot and the ratios were calculated. RESULTS: Compared with the blank group, the number and score of writhing, latency of writhing, pathological injury score, contents of PGF2α and ratios of PGF2α/PGE2 in the serum and uterine tissue, and the levels of p-PI3K/PI3K, p-Akt/Akt and p-mTOR/mTOR in the uterine tissue were significantly increased in the model group(P<0.01, P<0.05), while contents of PGE2 in the serum and uterine tissue were reduced(P<0.05). In comparison with the model group, the number of writhing and writhing score, pathological injury score, contents of PGF2α and ratios of PGF2α/PGE2 in both the serum and uterine tissue, the levels of p-PI3K/PI3K, p-Akt/Akt and p-mTOR/mTOR were obviously decreased(P<0.05, P<0.01), whereas the writhing latency was considerably prolonged in the EA group(P<0.01), with elevated contents of PGE2 in the serum and uterine tissue(P<0.05). H.E. staining showed slight dilation of uterine glandular cavity, and severe endometrial edema with extensive cell shedding and a large number of vacuole-like degeneration, apoptosis, pyknosis or fragmentation or disappearance of the nucleus, and neutrophil infiltration in the model group, which were relatively milder in the EA group. Ultrastructural results showed irregular fibroblasts of uterine tissue cells, obvious cytoplasmic edema, reduction in cytoplasmic electron density, seriously irregular nuclei, severe edema of mitochondria with dissolved matrix, fracture and disappearance of mitochondrial crests and vacuolation, and moderate dilation of rough endoplasmic reticulum in the model group, which were milder in the EA group. CONCLUSIONS: EA can improve pain and uterine inflammatory response in PDM rats, which may be associated with its functions in reducing uterine PGF2α and down-regulating PI3K/Akt/mTOR signaling.

[Effects of electroacupuncture on N-methyl-D-aspartate receptor and mitogen activated protein kinase in spinal cord of rats with primary dysmenorrhea].

OBJECTIVE: To observe the effects of electroacupuncture (EA) on the expression levels of N-methyl-D-aspartate receptor (NMDAR), extracellular signal-regulated kinase (ERK)1/2, p38 mitogen activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK) in the spinal cord of rats with primary dysmenoramia (PDM), so as to explore the underlying mechanism of EA treating PDM. METHODS: Thirty female SD rats were randomly divided into normal group, model group and EA group, with 10 rats in each group. The PDM rat model was established by subcutaneous injection of estradiol benzoate and oxytocin into the thigh. At the same time of modeling, rats in the EA group were treated with EA (50 Hz) at "Sanyinjiao" (SP36) and "Guanyuan" (CV4) once daily, 20 min each time, for 10 consecutive days. The writhing times, writhing score and writhing latency were observed within 30 min after oxytocin injection. The uterine pathological morphology was observed by HE staining, and pathological score was calculated. Serum prostaglandin F2α (PGF2α) and prostaglandin E2 (PGE2) were determined by ELISA. The protein expression levels of NMDAR, ERK1/2, p38MAPK and JNK in spinal cord were detected by Western blot. RESULTS: Compared with the normal group, the writhing times and writhing score were significantly increased (P<0.05); the endometrial epithelial cells showed vacuolar degeneration, death and hyperemia, the uterine pathological score was increased (P<0.05); the content of serum PGF2α and the ratio of PGF2α/PGE2 were significantly increased (P<0.01), while the content of serum PGE2 was significantly decreased (P<0.01); the expression levels of NMDAR, ERK1/2, p38MAPK and JNK in spinal cord were significantly increased (P<0.05, P<0.01) in the model group. Compared with the model group, the writhing times and writhing score were significantly decreased (P<0.05), the writhing latency was prolonged (P<0.05); the endometrial epithelial cells still showed vacuolar degeneration, death and hyperemia, and the uterine pathological score was decreased (P<0.01); the content of serum PGF2α and the ratio of PGF2α/PGE2 were significantly decreased (P<0.01), while the content of serum PGE2 was significantly increased (P<0.01); the protein expression levels of ERK1/2 and JNK in spinal cord were significantly decreased (P<0.01) in the EA group. CONCLUSION: EA intervention at SP36 and GV4 has obvious analgesic effect on PDM rats, and its mechanisms may be related to reducing serum prostaglandin, alleviating uterine inflammation, and inhibiting the protein expressions of NMDAR, ERK1/2, p38 MAPK and JNK in spinal cord.

[Effects of electroacupuncture on NLRP3 inflammasome and pyroptosis protein GSDMD in uterine tissue in rats with primary dysmenorrhea].

OBJECTIVE: To observe the effects of electroacupuncture (EA) on NLRP3 inflammasome and its downstream protein gastermin D (GSDMD) in rats with primary dysmenorrhea (PDM), and to explore the potential mechanism of EA on the treatment of PDM. METHODS: Forty healthy female SD rats without pregnancy were randomly divided into a control group, a model group, an EA group and an ibuprofen group, 10 rats in each group. PDM model was prepared by injection of estradiol benzoate and oxytocin. Except the control group, the rats in each group were subcutaneously injected with estradiol benzoate for 10 days, and oxytocin was injected on the 11th day. The rats in the EA group were intervened with EA (dense wave, frequency of 50 Hz) at "Guanyuan" (CV 4) and "Sanyinjiao" (SP 6) at the same time of modeling, once a day, 20 min each time, for 10 consecutive days. The rats in the ibuprofen group were treated with 0.8 mL of ibuprofen by gavage (concentration of ibuprofen solution was 1.25 mg/mL) for 10 consecutive days. After modeling, the writhing reaction was observed. After intervention, the HE staining method was used to observe the histological morphology of uterus and evaluate the pathological damage score of uterus; ELISA method was used to detect the serum levels of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α); Western blot method was used to detect the protein expression of NLRP3, apoptosis related spot like protein (ASC), caspase-1, GSDMD, GSDMD-N and inflammatory factors (interleukin [IL]-1ß, IL-18) in uterine tissue. RESULTS: In the model group, a large number of vacuolar degeneration and death of endometrial epithelial cells, spiral arterioles congestion in lamina propria and neutrophil infiltration were observed. In the EA group, there was a small amount of vacuolar degeneration and death of endometrial epithelial cells, a small amount of spiral arterioles congestion in the lamina propria, and a small amount of neutrophils infiltration. In the ibuprofen group, there was very small number of degeneration and death of endometrial epithelial cells, and no obvious arterial congestion was found in lamina propria, and neutrophil infiltration was occasionally seen. Compared with the control group, in the model group the number of writhing was increased (P<0.01), the writhing reaction score and serum level of PGF2α and PGF2α/PGE2 value were increased (P<0.01), the level of PGE2 was decreased (P<0.01). Compared with the model group, in the EA group and the ibuprofen group the number of writhing were decreased (P<0.05), the latency of writhing was prolonged (P<0.01), the writhing reaction scores and serum levels of PGF2α and PGF2α/PGE2 values were decreased (P<0.05, P<0.01), the levels of PGE2 were increased (P<0.01). Compared with the control group, the protein expression of NLRP3, ASC, caspase-1, GSDMD, GSDMD-N, IL-1ß and IL-18 in the uterine tissues of rats was increased in the model group (P<0.01). Compared with the model group, the protein expression of NLRP3, ASC, caspase-1, GSDMD, GSDMD-N, IL-1ß and IL-18 in the uterine tissues of rats was decreased in the EA group and the ibuprofen group (P<0.01, P<0.05). There was no significant difference between the EA group and the ibuprofen group in the above indexes (P>0.05). CONCLUSION: EA could alleviate pain and uterine tissue injury in rats with PDM. The mechanism may be related to the inhibition of the activation of NLRP3 inflammasome in rat uterine tissues, thereby inhibiting pyroptosis and its inflammatory factors release.

[Effect of electroacupuncture intervention on relieving pain and inflammation by suppressing TLR4/NF-κB signaling in rats with primary dysmenorrhea].

OBJECTIVE: To investigate the mechanism of electroacupuncture(EA) intervention in rats with primary dysmenorrhea(PDM) based on the Toll-like receptor 4(TLR4)/nuclear factor(NF)-κB signaling pathway. METHODS: Forty female SD rats were randomly divided into blank control, model, EA and medication groups, with 10 rats in each group. PDM rat model was established by subcutaneous injection of estradiol benzoate combined with intraperitoneal injection of oxytocin. At the same time of model procedures, EA(50 Hz, dense wave) was applied to "Guanyuan" (CV4) and bilateral "Sanyinjiao" (SP6) of rats in the EA group, with needles retained for 20 min, for 10 consecutive days. Rats in the medication group received ibuprofen(125 mg/100 mL, 0.8 mL) by gavage for 10 consecutive days. At the 11th day, writhing behavior of rats was assessed. Uterine morphology was observed by eyes and uterine pathological changes were observed after HE staining. Content of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) in serum and uterine tissues was detected by ELISA; NF-κB p65 positive expression in nucleus was detected by immunofluorescence; protein expression levels of TLR4, NF-κB p65, p-NF-κB p65 and inflammatory factors interleukin (IL) -1ß and IL-18 were detected by Western blot. RESULTS: After modeling, uterus tissues were congested and edematous, with necrosis of luminal epithelium, severe edema and extensive shedding of endometrium, nuclear pyknosis, fragmentation and disappearance, neutrophils infiltration, and slight expansion of glandular cavity, which was milder in the EA and the medication groups. Compared with the blank control group, writhing times, scores and incubation period, HE pathological scores, PGF2α contents in serum and uterine tissues, ratio of NF-κB p65 positive expression in nucleus, TLR4, NF-κB p65, p-NF-κB p65, IL-1ß and IL-18 protein expression levels in uterine tissues of rats in the model group were all significantly increased(P<0.01), while PGE2 contents in serum and uterine tissues were significantly decreased(P<0.01). Compared with the model group, writhing times and scores, HE pathological scores, PGF2α contents in serum and uterine tissues, ratio of NF-κB p65 positive expression in nucleus, TLR4, NF-κB p65, p-NF-κB p65, IL-1ß and IL-18 protein expression levels in uterine tissues of rats in the EA and medication group were all significantly decreased(P<0.01), while writhing incubation period, PGE2 contents in serum and uterine tissues were significantly increased(P<0.05, P<0.01). CONCLUSION: EA intervention could relieve inflammatory response and pain in PDM rats, which may be related to its effect in reducing TLR4 expression, inhibiting NF-κB activation and down-regulating inflammatory factors levels of IL-1ß and IL-18.

[Acupuncture at acupoints along the meridians for primary insomnia: a multi-center randomized controlled trial].

OBJECTIVE: To compare the clinical effect differences among different acupoint selection methods for primary insomnia. METHODS: A total of 333 patients with primary insomnia were recruited from 3 study centers and randomly divided into a group A (111 cases, 7 cases dropped off), a group B (111 cases, 5 cases dropped off) and a group C (111 cases, 2 cases dropped off). The patients in the group A were treated with acupuncture at Shenmen (HT 7) and Baihui (GV 20), the patients in the group B were treated with acupuncture at Sanyinjiao (SP 6) and Baihui (GV 20), and the patients in the group C were treated with acupuncture at non-acupoint and Baihui (GV 20). All the treatment was given once a day, 30 min each time; 5 treatments were taken as a course and 5 courses of treatment were given. The Pittsburgh sleep quality index (PSQI) and Athens insomnia scale (AIS) scores were evaluated before and after treatment as well as 4 weeks after treatment. The encephalofluctuograph technology (ET) was observed before and after treatment. RESULTS: Compared before treatment, the PSQI scores after treatment and at follow-up were significantly decreased in three groups (P<0.01), and the decrease in the group A and the group B was greater than that in the group C (P<0.01). Compared before treatment, the AIS scores after treatment and at follow-up was significantly decreased in three groups (P<0.01), and the decrease in the group A was greater than that in the group C (P<0.05). The interclass and between-groups ET before and after treatment had no significant difference (P>0.05). CONCLUSION: The acupuncture at acupoints along the meridians could improve the sleep quality in patients with primary insomnia, and the therapeutic effect of acupoint along the meridians is better than that of non-acupoint.

[Manual Acupuncture Stimulation of Paired Acupoints Can Relieve Sleep Disorder Possibly by Upregulating Pineal Melatonin Protein and Its Receptor mRNA Levels in the Suprachiasmatic Nucleus in Insomnia Rats].

OBJECTIVE: To observe the effect of manual acupuncture stimulation of paired "Baihui" (GV 20)+ "Shenmen" (HT 7), GV 20+ "Sanyinjiao" (SP 6), and GV 20+ non-acupoint on expression of melatonine (MT) and suprachiasmatic melatonin receptor 1 (MT1) and melatonin receptor 2 (MT2) mRNAs in insomnia rats, so as to explore their action difference and the underlying mechanism in improving insomnia. METHODS: Male SD rats were randomly divided into normal control (n=12), mo-del (n=8), GV 20+HT 7(n=12), GV 20+SP 6(n=11), and GV 20+ non-acupoint (n=10) groups. The insomnia model was established by intraperitoneal injection of Para-chlorophenylalanine suspension (50 mg/mL, 50 mg/100 g), once daily for 2 days. The abovementioned acupoints GV 20, bilateral HT 7, SP 6 and non-acupoints (the midpoint between the elbow-tip and armpit on the medial side of the upper-arm) were punctured with filiform needles and manipulated by rotating the needle for about 1 min which was repeated once again every 10 min during 30 minutes' needle-retaining. The treatment was conducted once daily for 7 days. The expression levels of MT immunoactivity in the conarium tissue, and MT1 and MT2 mRNAs of the suprachiasmatic nucleus (SCN) region were detected using immunohistochemistry and fluorescence quantitative real time-PCR, respectively. RESULTS: After modeling, the expression levels of pineal MT (an increase of gray value means a decrease of immunoactivity), SCN MT1 and MT2 mRNAs were notably down-regulated in the model group relevant to the normal control group (P<0.05, P<0.01). Following the treatment, the down-regulated expression levels of MT protein, and MT1 and MT2 mRNAs were obviously reversed in the GV 20 + HT 7, GV 20 + SP 6 groups relevant to the model group (P<0.05, P<0.01). The therapeutic effect of GV 20+ HT 7 was superior to that of GV 20+ non-acupoint in up-regulating the expression of MT1 mRNA (P<0.01), and markedly superior to that of GV 20+ SP 6 and GV 20+ non-acupoint in increasing the sleep duration and in up-regulating the expression of MT2 mRNA (P<0.01). No significant differences were found among the 3 treatment groups in up-regulating the expression of MT (P>0.05). CONCLUSION: Manual acupuncture stimulation of GV 20+ HT 7 and GV 20+ SP 6 can improve the sleep disorder in insomnia rats, which may be related to its effects in increasing the levels of pineal MT protein, and MT1 and MT2 mRNAs in hypothalamic SCN.

[Effect of Different Stimulating Strength of Electroacupuncture on Gastrointestinal Motility and RhoA/ROCK Signaling in Gastric Antral Smooth Muscle in Diabetic Gastroparesis Rats].

OBJECTIVE: To observe the effect of different strength of electroacupuncture (EA) stimulation on gastrointestinal motility and Ras homolog gene family member (RhoA)/Rho associated coiled-coil forming protein kinase (ROCK) signaling in diabetic gastroparesis (DGP) rats, so as to reveal the underlying mechanisms of EA for improving DGP. METHODS: Sixty SD rats were randomly and equally divided into blank control, DGP model, weak EA, medium EA, and strong EA groups (n＝12 rats in each). The DGP model was established by intraperitoneal injection of streptozotocin (STZ, 55 mmol/kg, 2%) and high-sugar and high-fat fodder feeding for 8 weeks. EA (0.12, 0.24, 0.36 mA, 20 Hz/100 Hz) was applied to "Zusanli" (ST 36), "Sanyinjiao" (SP 6) and "Liangmen" (ST 21) for 20 min, once daily for 15 successive days. Blood glucose levels were measured weekly with blood glucose meter and blood glucose test paper. Fecal phenol red excretion method was used to display gastric emptying and small intestinal propulsion function. The expression of RhoA protein in the gastric antral smooth muscle tissue was detected by immunohistochemistry and Western blot (WB), separately, and that of ROCK, myosin phosphatase target subunit 1 (MYPT 1) and phosphorylated (p)-MYPT 1 proteins in gastric antrum detected by WB. RESULTS: Compared with the blank control group, the gastric emptying rate and small intestine propulsion rate of the model group were significantly decreased (P<0.05), and the blood glucose level was remarkably increased (P<0.05). Moreover, the expression levels of RhoA, ROCK, MYPT 1 and p-MYPT 1 proteins in the gastric antrum were significantly down-regulated relevant to the control group (P<0.05). After administration of EA, the decreased gastric emptying rate and intestinal propulsion rate, and the down-regulated expression of RhoA, ROCK, MYPT 1 and p-MYPT 1 proteins were significantly increased in the strong, medium and weak EA stimulation groups (P<0.05). Comparison among the 3 EA groups showed that the strong stimulation was significantly superior to weak stimulation in up-regulating the expression of RhoA, ROCK, MYPT 1 and p-MYPT 1 proteins, and obviously superior to the medium stimulation in up-regulating RhoA and MYPT 1 protein levels (P<0.05), while the medium stimulation was significantly stronger than the weak stimulation in up-regulating the expression of ROCK, MYPT 1 and p-MYPT 1 proteins (P<0.05). There were no significant differences among the 3 EA groups in up-regulating the gastric emptying rate and small intestinal propulsion rate, and between the strong stimulation and medium stimulation in the expression levels of ROCK and p-MYPT 1 proteins (P>0.05). CONCLUSION: Electroacupuncture stimulation of ST 36-SP 6-ST 21 at 0.12, 0.24 and 0.36 mA can promote the gastrointestinal motility in DGP rats, which may be associated with its effects in enhancing RhoA/ROCK signaling in the gastric antral smooth muscle at different degrees.

[Effects of Herbal-cake-separated Moxibustion on Blood Lipid Protein Levels and Expression of Toll-like Receptor and Nuclear Factor Genes in Atherosclerotic Plaques in Hyperglycemia Rabbits].

OBJECTIVE: To observe the effect of herbal cake-separated moxibustion on blood lipid-apoprotein levels and the expression of Toll-like receptor 2 (TLR 2), TLR 4 and nuclear factor kappa B(NF-κB) mRNAs in atherosclerotic (AS) vulnerable plaques of hyperglycemia rabbits, so as to explore its mechanism underlying improvement of atherosclerosis. METHODS: Sixty New Zealand rabbits were randomly divided into 5 groups: control, model, direct moxibustion, herbal-cake-separated moxibustion and medication groups(n＝12 rabbits in each group). The AS vulnerable plaque model was established by high-fat forage feeding plus balloon-induced abdominal aorta injury and gene transfection of Ad 5-p 53 recombinant vector. Direct moxibustion or herbal-cake-separated moxibustion was applied to "Juque" (CV 14) and bilateral "Tianshu" (ST 25), "Fenglong" (ST 40), or bilateral "Xinshu" (BL 15), "Pishu" (BL 20) and "Ganshu" (BL 18) for 15－20 min every time. The medication group was treated by feeding Atorvastatin. All the treatments were conducted once daily for 8 weeks. Plasma total cholesterol(TC) and triglyceri-de(TG) contents were detected by enzyme method, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) contents detected by colorimetric determination, and plasma apolipoprotein A(Apo-A) and apolipoprotein B(Apo-B) levels determined by electrophoretic method. The pathological changes of vulnerable plaque and the aortic intima and media thickness were observed under light microscope after H．E. staining. The expression levels of TLR 2, TLR 4 and NF-κB mRNAs in AS plaques were determined by quantitative real-time PCR. RESULTS: After modeling, the levels of plasma TC, TG, LDL and Apo-B in the model group were remarkably increased (P<0.01), and Apo-A and HDL/LDL were significantly decreased in comparison with the control group (P<0.01). Additionally, the aortic intima and media thickness and the expression levels of TLR 2, TLR 4 and NF-κB mRNAs in AS plaques were significantly increased (P<0.01). After the treatment, the elevated levels of plasma TC, TG, LDL and Apo-B, the aortic intima thickness and media thickness, and the expression levels of TLR 2, TLR 4 and NF-κB mRNAs in the 3 treatment groups were significantly down-regulated in comparison with the model group (P<0.05, P<0.01), while the decreased levels of Apo-A and HDL/LDL were considerably increased (P<0.01). Comparison among the 3 treatment groups showed that the therapeutic effects of the herbal-cake-separated moxibustion and medication were significantly superior to those of the direct moxibustion in down-regulating the levels of TC, TG, LDL, Apo-B, TLR 2 mRNA, TLR 4 mRNA and NF-κB mRNA, and reducing the thickness of the aortic intima and media, as well as in up-regulating the levels of Apo-A and HDL/LDL (P<0.05, P<0.01). No significant differences were found between the herbal-cake-separated moxibustion and medication groups in the above-mentioned indexes (P>0.05). CONCLUSION: Herbal-cake-separated moxibustion has a positive role in stabilizing AS vulnerable plaque in hyperglycemia rabbits, which may be associated with its effects in regulating blood lipid-apolipoprotein levels and inhibiting the expression of TLR 2, TLR 4 and NF-κB mRNAs in vulnerable plaques.

[Effect of Acupuncture Stimulation of Different Acupoint Combinations on Sleep and Expression of Circadian Clock and Bmal 1 Genes in Hypothalamus of Insomnia Rats].

OBJECTIVE: To observe the effect of manual acupuncture stimulation (MAS) of "Baihui"(GV 20)-"Shenmen"(HT 7)or GV 20-"Sanyinjiao"(SP 6) on sleep and expression of circadian rhythm genes (Clock and Bmal 1) in the hypothalamus in insomnia rats, so as to select a better acupoint group for insomnia. METHODS: A total of 53 SD rats were randomly divided into normal control (n=12),insomnia model (n=8), GV 20-HT 7(n=12), GV 20-SP 6(n=11),and GV 20-non-acupoint (n=10) groups. The insomnia model was established by intraperitoneal injection of p-chlorophenylalanine (PCPA,500 mg/kg,100 mg/mL) once daily for 2 days. The MAS (uniform reinforcing-reducing needling) was applied to GV 20-HT 7, GV 20-SP 6 or GV 20-non-acupoint for 30 min,once daily for 7 days. The sleep onset latency and sleep duration were gauged after intraperitoneal injection of pentobarbital sodium (35 mg/kg). The expression levels of Clock mRNA and Bmal 1 mRNA in the hypothalamic tissues containing ventrolateral preoptic area (VLPO) and suprachiasmatic nucleus (SCN) region were detected by fluorescence quantitative real-time PCR. RESULTS: Following administration of pentobarbital sodium,the sleep latency was significantly prolonged and the sleep duration was considerably shortened in rats of the model group(P<0.05). After the treatment, the increased sleep latencies in the GV 20-HT 7, GV 20-SP 6 and GV 20-non-acupoints were all significantly down-regulated (P<0.05), and the decreased sleep duration was significantly increased only in the GV 20-HT 7 group relevant to the model group (P<0.05), but not in the GV 20-SP 6 and GV 20-non-acupoint groups (P<0.05). There were no significant differences in the sleep latency among the 3 treatment groups (P<0.05). The sleep duration was obviously prolonged in the GV 20-HT 7 group than in the GV 20-SP 6 and GV 20-non-acupoint groups (P<0.05). After modeling, the expression levels of Clock mRNA and Bmal 1 mRNA in hypothalamic VLPO and SCN regions were significantly down-regulated relevant to the normal control group (P<0.01). Following the treatment, the expression levels of Clock mRNA in the VLPO and SCN regions of the GV 20-SP 6 and GV 20-HT 7 groups, and those of Bmal 1 mRNA in the VLPO and SCN regions of the 3 treatment groups were considerably increased relevant to the model group (P<0.05, P<0.01). The effects of GV 20-HT 7 were significantly superior to those of GV 20-SP 6 and GV 20-non-acupoint (and also the action of GV 20-SP 6 was evidently superior to that of GV 20-non-acupoint) in up-regulating the expression of Clock mRNA and Bmal 1 mRNA in both VLPO and SCN regions (P<0.05, P<0.01). CONCLUSIONS: Manual acupuncture stimulation of GV 20-HT 7 can improve the sleep latency and duration in insomnia rats,which may be associated with its effects in up-regulating the expression levels of circadian Clock mRNA and Bmal 1 mRNA in hypothalamic VLPO and SCN regions, and the efficacy of GV 20-HT 7 is obviously better than that of GV 20-SP 6 and GV 20-non-acupoint.

[Effect of Point-moxibustion and Electroacupuncture on the Expression of Endothelial Nitric Oxide Synthase mRNA and Angiotensin 2 mRNA in Gastric Antrum in Diabetic Gastroparesis Rats].

OBJECTIVE: To observe the effect of point-moxibustion and electroacupuncture(EA) on gastrointestinal motility, and expression of endothelial nitric oxide synthase(eNOS) mRNA and angiotensin 2(AT Ⅱ) mRNA in gastric antrum in diabetic gastroparesis(DGP) rats, so as to reveal the different effect between point-moxibustion and EA, and explore the mechanism underlying improvement of DGP. METHODS: Forty SD rats were randomly divided into normal, model, point-moxibustion and EA groups, 10 rats in each group. The DGP model was established by intraperitoneal injection of streptozotocin (STZ, 2%, 55 mmol/kg) and fed with high-sugar and high-fat fodder. "Zusanli" (ST 36), "Sanyinjiao" (SP 6) and "Liangmen" (ST 21) were chosen to be stimulated with moxibustion at 0, 10 and 20 min in the point-moxibustion group or with EA for 20 min in the EA group. The treatment was conducted once daily for 15 days. After treatment, blood glucose, gastric emptying rate and small intestinal propulsive rate were measured. The plasma endothelin 1(ET-1) content was detected by ELISA. The mRNA expression of eNOS and AT Ⅱ in gastric antrum were detected by Real-time PCR. RESULTS: Compared with the normal group, the blood glucose in the model group was significantly higher (P<0.01). Compared with the model group, the blood glucose decreased significantly in the point-moxibustion group and EA group (P<0.05). In comparison with the normal group, gastric emptying rate and small intestinal propulsive rate were decreased, the content of plasma ET-1 was increased, the expression of eNOS mRNA in gastric antrum was significantly reduced and the expression of ATⅡ mRNA was significantly increased in the model group(P<0.01). In comparison with the model group, gastric emptying rate and small intestinal propulsive rate were increased, the content of plasma ET-1 was decreased, the expression of eNOS mRNA in gastric antrum was higher and the expression of AT Ⅱ mRNA was significantly lower in both point-moxibustion group and EA group (P<0.05), and there were no significant differences between the two treatment groups in above mentioned indexes (P>0.05). CONCLUSIONS: Both point-moxibustion and EA can effectively promote gastrointestinal motility in DGP rats and improve the symptoms of delayed gastric emptying. The two therapies are effective, and their mechanism may be related to the increase of eNOS mRNA expression and the decrease of AT Ⅱ mRNA expression in gastric antrum.

[Effects of Electroacupuncture on Ultrastructure of Interstitial Cells of Cajal and Stem Cell Factor-kit Signal Pathway of Gastric Antrum in Diabetic Gastroparesis Rats].

OBJECTIVE: To observe the effects of electroacupuncture (EA) on gastrointestinal motility and the ultrastructure of interstitial cells of Cajal (ICC) and the expressions of c-kit receptor protein and stem cell factor (SCF) mRNA in diabetic gastroparesis (DGP) rats, so as to explore its mechanism. METHODS: Fifty SD rats were randomly divided into normal, model, acupoint, non-acupoint and metoclopramide groups (n＝10 rats/group). DGP model was established by intraperitoneal injection of streptozotocin (STZ, 2%), and raised with high-sugar high-fat diet irregularly. EA (sparse-dense, 10 Hz/50 Hz, 2 mA, 20 min) was applied at "Zusanli" (ST 36), "Liangmen" (ST 21) and "Sanyinjiao" (SP 6), and the corresponding non-acupoints of the 3 acupoints, daily for 15 days. The rats in metoclopramide group received intragastric administration of metoclopramide (1.7%, 1 mL/100 g) for 15 days, once a day. Blood sugar was determined with One Touch blood glucose test paper. The gastric emptying rate (GER) and the intestinal propulsion rate (IPR) were measured by intragastric phenol red. The ultrastructure of ICC was detected by transmission electron microscopy. The expression levels of c-kit receptor protein and SCF mRNA of gastric antrum were examined respectively by Western blot and RT-PCR. RESULTS: Compared with the normal group, the blood glucose significantly increased in the model group (P<0.01), while the GER, IRP and the expression level of SCF mRNA in the gastric antrum significantly decreased (P<0.01), and the ultrastructure of ICC appeared apoptosis-like changes. The blood glucose of the EA group was obviously decreased compared with that of the model group (P<0.05); the GER and IRP significantly increased(P<0.05, P<0.01); the expression level of SCF mRNA increased (P<0.01), the number of ICC increased and its ultrastructure was repaired. There was some relief on ICC ultrastructure in the acupoint group compared with that in the non-acupoint group; and SCF mRNA increased (P<0.05). There was no significant difference on c-kit receptor expression among all the modeling groups (P＞0.05). CONCLUSIONS: EA at ST 36, etc. can regulate the blood glucose and improve gastrointestinal emptying in DGP rats. The mechanism may be related to up-regulating SCF mRNA, repairing ICC ultrastructure, restoring the pacing function, and improving gastrointestinal motility.

[Clinical evaluation study on long-term effect of acupuncture with pattern/syndrome differentiation on functional dyspepsia].

OBJECTIVE: To compare the efficacy difference in the treatment of functional dyspepsia between acupuncture at the acupoints selected by pattern/syndrome differentiation and domperidone. METHODS: Seventy cases were randomized into an acupuncture group (35 cases) and a western medication group (35 cases). In the acupuncture group, Zusanli (ST 36) and Neiguan (PC 6) were selected. Taichong (LR 3) and Neiting (ST 44) were added for excess syndrome while Gongsun (SP 4) and Yinlingquan (SP 9) were added for deficiency syndrome. A pair of electrodes was attached to one acupoint and an assistant point (2 mm next to the acupoint centripetally) and stimulated with disperse-dense wave at 2 Hz/100 Hz, once a day. In the western medication group, domperidone was prescribed for oral administration, 10 mg each time, three times a day. In the two groups, the treatment of 5 days made one session and 4 sessions were required totally. Nepean dyspepsia index (NDI) was compared after treatment, 1, 2, 3, 4 and 5 months after treatment between the two groups respectively. RESULTS: The score of symptom and score of life quality in NDI after treatment and at each follow-up time point were improved obviously in the acupuncture group as compared with those before treatment (all P < 0.01). In the western mediation group, the score of symptom and the score of life quality in NDI after treatment and in follow-up of 1, 2 and 3 months were improved obviously as compared with those before treatment (all P < 0.01), but the differences were not significant in follow-up of 4 and 5 months (both P > 0.05). Compared with the western medication group, the symptom score of NDI was reduced obviously after treatment and in each time point of follow-up in the acupuncture group (P < 0.05, P < 0.01), and the score of life quality was increased obviously (P < 0.05, P < 0.01). CONCLUSION: Acupuncture at the acupoints selected by pattern/syndrome differentiation and domperidone are effective in the treatment of functional dyspepsia. Domperidone is unsatisfactory in the long-term effect, but acupuncture achieves the positive short-term and long-term effects on functional dyspepsia.

[Clinical observation on therapeutic effects of mild moxibustion in superficial gastritis patients with spleen-stomach deficiency-cold pattern].

OBJECTIVE: To observe the curative effect of different duration of mild moxibustion in superficial gastritis patients with deficiency cold pattern of spleen and stomach. METHODS: A total of 84 superficial gastritis patients were randomly divided into moxibustion-20 min group, moxibustion-40 min group and medication (Ranitidine) group, with 28 cases in each group. Mild moxibustion was applied to bilateral Zusanli (ST 36) and Zhongwan (CV 12, odd days), Pishu (BL 20) and Weishu (BL 21, even-numbered days) for 20 min and 40 min respectively, once daily for 20 days. The symptom integrative score of superficial gastritis patients and the therapeutic effects were assessed according to "The Scoring Table for Symptoms and Signs of Gastrointestinal Disorders of Chinese Medicine", etc. RESULTS: Following 20 days' treatment, of the three 28 gastrititis patients in the medication, moxibustion-20 min and moxibustion-40 min groups, 0, 1, and 0 cases were cured, 5, 7 and 12 experienced marked improvement in their symptoms, 15, 16 and 12 had an improvement, and 8, 4 and 4 were invalid, with the effective rates being 71.4%, 85.7% and 85.7%, respectively. After one month's follow up, of the three 28 cases in the medication, moxibustion-20 min and moxibustion-40 min groups, 0, 4 and 0 cases were cured, 4, 8, and 13 experienced marked improvement, 15, 16 and 13 had an improvement, 9, 0 and 2 were ineffective, with the effective rates being 67.9%, 100% and 92.9%, respectively. Self-comparison of each group showed that the integrative scores of symptoms were all decreased significantly after the treatment and one-month's follow up in the three groups (P < 0.01). The effective rates of both moxibustion-20 min and -40 min were significantly higher than that of the medication group after one month's follow up (P < 0.01, P < 0.05). The therapeutic effect of moxibustion-20 min group was significantly superior to that of the medication group in lowering the integrative score of symptoms after one month's follow up (P < 0.01). No significant differences were found among the three groups in the therapeutic effects and in reducing integrative scores of symptoms after 20 days' treatment (P > 0.05). CONCLUSION: Mild moxibustion is significantly superior to medication in long-term therapeutic effects in the treatment of superficial gastritis patients with spleen-stomach deficiency-cold pattern.

[Comparative study on effects between electroacupuncture and acupuncture for spastic paralysis after stroke].

OBJECTIVE: To observe the differences of therapeutic effect of spastic paralysis after stroke between electroacupuncture and acupuncture and explore the mechanism. METHODS: Sixty-four cases were randomly divided into an electroacupuncture group (n = 33) and an acupuncture group (n = 31). Both groups were treated with Bobath facilitation techniques and medicine treatments. Quchi (LI 11), Hegu (LI 4), Yanglingquan (GB 34), Sanyinjiao (SP 6), et al. on the affected side were selected in each group. The needle was retained for 30 min, and the manipulation was applied for 1 min in the acupuncture group, and electroacupuncture was added in the electroacupuncture group. Stroke Impairment Assessment Set (SIAS) was adopted to assess the whole function status after sroke, and the contents of glutamate (Glu) and gamma-aminobutyric acid (GABA) in serum and clinical efficacy were observed in the two groups. RESULTS: The SIAS score increased after treatment as compared with that before treatment in either group (both P < 0.01), and the electroacupuncture group was superior to the acupuncture group (P < 0.01); the content of Glu in blood serum and ratio of Glu/GABA reduced, while the content of GABA in serum increased after treatment as compared with those before treatment in either group (all P < 0.01), but the improvement of above indices were much more apparently in the electroacupuncture group as compared with those in the acupuncture group (P < 0.01, P < 0.05); the total effective rate of 90.9% (30/33) in the electroacupuncture group was superior to that of 83.9% (26/31) in the acupuncture group (P < 0.05). CONCLUSION: Electroacupuncture can improve therapeutic effect of spastic paralysis after stroke, it's mechanism may be ralated to ajusting the contents of Glu and GABA in serum.

The effect of herb-partition moxibustion on Toll-like receptor 4 in rabbit aorta during atherosclerosis.

OBJECTIVE: To explore the mechanism of Toll-like receptor (TLR4) inhibition in the delay of formation of atherosclerosis by herb-partition moxibustion. METHOD: Seventy-five rabbits were randomly assigned to one of five groups: blank, atherosclerosis (AS) model, direct moxibustion, herb-partition moxibustion, and drug treatment. With the exception of the blank group, all rabbits were given a high-fat diet in addition to immunologic injury to create the AS model. The experiments were carried out for 16 weeks, at which time the aorta was removed from each rabbit. Immunohistochemical methods were used to detect the gray level of the aortic TLR4 to observe the immunologic competence of its antigens. Fluorescent quantitative polymerase chain reaction was used to detect the expression of TLR4 messenger RNA (mRNA) in the aorta. RESULTS: The gray-scale value of TLR4 and the TLR4 mRNA expression significantly decreased (p<0.05) in the direct moxibustion, herb-partition moxibustion, and drug treatment groups. Furthermore, the effects of the herb-partition moxibustion and drug treatment were superior to those of the direct moxibustion. CONCLUSION: Herb-partition moxibustion inhibits aortic TLR4 activity and mRNA expression, showing that herb-partition moxibustion delays the formation of atherosclerosis through the inhibition of TLR4 expression.

[Effects of moxibustion on gastric mucosal EGF and TGF-alpha contents and epidermal growth factor receptor expression in rats with gastric mucosal lesion].

OBJECTIVE: To observe the effects of moxibustion on contents of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) and epidermal growth factor receptor (EGFR) expression in the gastric mucosa tissue in rats with gastric mucosal lesion. METHODS: Thirty-six SD rats were randomly and equally divided into control, model and moxibustion groups. Gastric mucosal lesion model was duplicated by restraint and cool water immersion stress. Pre-moxibustion was applied to "Zusanli" (ST 36) and "Zhongwan" (CV 12), "Pishu" (BL 20) and "Weishu" (BL 21) alternately, once everyday for 8 days before modeling. The contents of EGF and TGF-alpha in gastric mucosa were detected by enzyme-linked immunosorbent assay (ELISA) and the expression of EGFR determined by immunohistochemistry. RESULTS: Compared with the control group, only TGF-alpha content in the gastric mucosa in the model group was increased significantly (P < 0.05). Compared with the model group, the EGF and TGF-alpha contents and EGFR immunoactivity in the gastric mucosa were increased significantly in the moxibustion group (P < 0.05, P < 0.01). CONCLUSION: Pre-moxibustion at ST 36, CV 12, BL 20 and BL 21 can up-regulate gastric mucosal EGF and TGF-alpha contents and EGFR protein expression in gastric mucosa lesion rats, which may contribute to its effect in relieving stress-induced gastric mucosal injury.

[On needle-sticking method].

Needle-sticking method has essential differences from stuck needle induced by acupuncture accident. This manipulation refers to the needle-sticking manifestation induced by twirling the needle in one direction after arrival of qi so as to tangle muscle fibers, which can combined with some compound methods such as trembling, shaking, flying, lifting, plucking, dragging and so on. It is effective for excessive syndrome, pain syndrome, arthralgia syndrome, etc. and with functions of promoting flow of qi and inducing qi to carry out stimulating circulation of channel-qi, promoting the needling sensation propagating along the channel and accelerating qi reaching to the affected region. Its main adverse reactions are pain, tissue damage and so on. The selection of needling instruments, the needling depth, the twirling intensities and location of forbidden or careful application must be paid attention in concrete practice.

[Observation on therapeutic effect of opposing needling for treatment of poststroke shoulder-hand syndrome].

OBJECTIVE: To compare the therapeutic effects of opposing needling and routine acupuncture for treatment of poststroke shoulder-hand syndrome. METHODS: Sixty cases were randomly divided into an opposing needling group and a routine acupuncture group, 30 cases in each group. In the two groups, Jianyu (LI 15), Jianliao (TE 14), Quchi (LI 11), Hegu (LI 4), etc. were selected, with on the healthy side selected for the opposing needling group and on the affected side selected for the routine acupuncture group. The motor function of the affected limb was evaluated by Fugl-Meyer scale, activity of daily living by ADL scale, and pain by VAS, and the edema degree was investigated before and after treatment. RESULTS: There were significant differences before treatment and after 2 therapeutic courses in the scores of Fugl-Meyer for the upper limb motor function and the ADL score for activity of daily living (both P<0.05), with the opposing needling group being significantly better than the routine acupuncture group (P<0.05); after treatment, both the edema degree and the pain scores significantly decreased (both P<0.05), with the opposing needling group in improvement of the edema degree being better the routine acupuncture group (P<0.05), and with no significant difference between the two groups in improvement of pain (P>0.05); the total effective rate was 93.3% in the opposing needling group and 90.0% in the routine acupuncture group with a significant difference between the two groups (P<0.05). CONCLUSION: The clinical therapeutic effect of the opposing needling is better than that of routine acupuncture therapy for treatment of poststroke shoulder hand syndrome.

[Effects of cake-separated moxibustion on ultrastructures of endothelial cells of aorta in the rabbit of hyperlipemia].

OBJECTIVE: To explore the mechanism of cake-separated moxibustion in treatment of hyperlipemia. METHODS: Sixty rabbits were randomly divided into 4 groups, a blank group,a model group, a direct moxibustion group and a cake-separated moxibustion group. Hyperlipemia model was developed by high fat diet of cholesterol. Changes of ultrastructures of endothelial cells of the aorta of the rabbit were observed with electron microscope. RESULTS: The endothelial cells in the cake-separated moxibustion group were more intact, most of them were normal in forms, internal elastic membrane was continuous, their thickness was even, the cells of smooth muscles in the medial membrane were relatively normal, which are similar to those in the blank control group. But the structure of endothelial cells of the aorta in the model group disappeared, in cytoplasm the sedimentation of a great number of lipids can be seen, internal elastic membrane was interrupted, the thickness was uneven, with focal dissolution, the cells of smooth muscle in the medial membrane had sedimentation of lipids, with frothy change. CONCLUSION: Cake-separated moxibustion has a certain protective action on endothelial cells of the aorta in the rabbit of hyperlipemia.