[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.