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-nonacupoint 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 realtime 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-nonacupoint 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).Conclusion 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.