| Purpose:It was believed that acupuncture at “Tsusanli” had the function of replenishing the Spleen-Stomach Qi,improving the ability of stomach meridian Qi and blood circulation as early as the Jin and Yuan Dynasties.In this experiment,forty SD rats were randomly divided into four groups: normal group,Spleen Qi deficiency group,Tsusanli group and non acupoint group.The model of Spleen Qi deficiency syndrome was established by the composite methods of improper diet and overstrain except the normal group.The rats were treated with electroacupuncture at the bilateral "Tsusanli" and non-acupoint intervention seven days after the model was successfully established.The gene and protein expression of brain-gut peptide related to energy metabolism in hypothalamus and small intestine of rats were detected by Q-PCR and ELISA.The gene and protein expression of brain-gut peptide related to energy metabolism in hypothalamus and small intestine of rats were detected by Q-PCR and ELISA.The changes of NRF1 and NRF2 mRNA and protein expression in gastric and skeletal muscle tissues of rats were detected by Q-PCR and WB.The purpose of this study was to explore the regulation mechanism of electroacupuncture at bilateral Zusanli points on Spleen Qi deficiency rats from the perspective of energy metabolism,providing theoretical basis for clinical acupuncture treatment.Materials and methods:Forty SD rats were divided into normal group,Spleen Qi deficiency group,Tsusanli group and non-acupoint group according to random number table(n=10).After all rats were adaptable for seven days in our animal experimental center,rats in the normal group drank water daily,the model of Spleen Qi deficiency syndrome was established by the composite methods of improper diet and overstrain except the normal group.Rats were normally reared,and electroacupuncture intervened between Tsusanli group and non-acupoint group when the Spleen Qi deficiency rat model was successfully constructed which conforms to the evaluation standard of Spleen Qi deficiency syndrome in Experimental Animal Model Methodology of Traditional Chinese Medicine,as the symptoms of anorexia(loss of appetite),fatigue,discoloration,leanness and drowsiness were observed in rats after five cycles.The rats were treated with electroacupuncture at the bilateral "Tsusanli" in the Tsusanli group,and the non-acupoint group were treated withelectroacupuncture bilateral non-acupoint intervention(Selection of a fixed control point in a10-15 mm posterior median line adjacent to the 20 mm segment of bilateral iliac crest was determined as a non-meridian acupoint point),the normal group and Spleen Qi deficiency group were fixed on the operating table with no other treatment after the model was successfully established.Acupuncture methods: the filiform needle was directly punctured for5 mm,followed by Huatuo Brand Electroacupuncture Instrument(SDZ-II).Dense wave stimulation(dense wave 15 Hz,sparse wave 2Hz)was used,and the current was 0.5mA,1time/ day,20min/time.All rats were fasting for 24 hours after 7 days of treatment and anesthetized with 10% chloral hydrate(3ml/kg body weight)the next day.Gastric and small intestinal tissues were cleaned with PBS solution after abdominal operation,hypothalamus tissue was taken off,the quadriceps femoris muscle tissues were obtained after cutting the skin of the hind limbs of rats.All tissues were frozen in liquid nitrogen and stored in-80?refrigerator for storage.The changes of Ghrelin,VIP,and CCK protein in small intestine and hypothalamus were detected by enzyme linked immunosorbent assay.The expression of Ghrelin,VIP,CCK and its receptor mRNA in small intestine and hypothalamus,and the expression levels of NRF1 and NRF2 mRNA in gastric and skeletal muscle tissues were detected by fluorescence quantitative PCR method.Changes of NRF1 and NRF2 protein contents in rat stomach and skeletal muscle tissues were detected by Western Blot.The experimental data were analyzed by SPSS 19.0 statistical software.The data were tested by normality test and variance homogeneity test,and measurement data with mean ±standard deviation,said the groups were analyzed using ANOVA analysis.It is considered that there is statistical difference when P?0.05.Results:1.Study on the content of brain-gut peptide and its receptor's mRNA in rats of each group1.1 Ghrelin protein content,gene expression and its receptor gene expression in rats of different groupThe results showed that compared with the normal group,the content of Ghrelin in small intestine decreased(P?0.05),the mRNA expression of Ghrelin and its receptor in small intestine and hypothalamus both decreased(P?0.05)in Spleen Qi deficiency group andnon-acupoint group;Compared with Spleen Qi deficiency group,the content of Ghrelin in small intestine,the mRNA expression of Ghrelin and its receptor in small intestine and hypothalamus both increased in Tsusanli group(P?0.05);There was no significant difference between Spleen Qi deficiency group and non-acupoint group(P?0.05).1.2 VIP protein content,gene expression and its receptor gene expression in rats of each groupCompared with the normal group,the content of VIP in small intestine decreased(P?0.05),the mRNA expression of VIP and its receptor in small intestine and hypothalamus both decreased(P?0.05)in Spleen Qi deficiency group and non-acupoint group;Compared with Spleen Qi deficiency group,the content of VIP in small intestine,and mRNA expression of VIP and its receptor in small intestine and hypothalamus both increased in Tsusanli group(P?0.05);There was no significant difference between Spleen Qi deficiency group and non-acupoint group(P?0.05).1.3 CCK protein content,gene expression and its receptor gene expression in rats of different groupCompared with the normal group,the content of CCK in small intestine increased(P?0.05),the mRNA expression of CCK and its receptor in small intestine and hypothalamus both increased(P?0.05)in Spleen Qi deficiency group and non-acupoint group;Compared with Spleen Qi deficiency group,the content of CCK in small intestine,the mRNA expression of CCK and its receptor in small intestine and hypothalamus both decreased in Tsusanli group(P?0.05);There was no significant difference between Spleen Qi deficiency group and non-acupoint group(P?0.05).2.Gene and protein expression of NRF1 and NRF2 in gastric and skeletal muscles of rats in each groupCompared with the normal group,the protein expression of NRF1 and NRF2,its mRNA in stomach and skeletal muscle tissue of rats were both significantly decreased in Spleen Qi Deficiency and non-acupoint group(P?0.05);Compared with the Spleen Qi deficiency group,the protein expression of NRF1 and NRF2 in stomach and skeletal muscle tissue of rats with Tsusanli group was significantly increased,as well as the mRNA expression of NRF1 and NRF2(P?0.05).There was no significant difference between Spleen Qi deficiency group andnon-acupoint group(P?0.05).Conclusion:1.Electroacupuncture at “Tsusanli” acupoint exerts its peripheral and central effects on regulating spleen-qi deficiency syndrome from the point of energy metabolism by regulating the expression of Ghrelin,VIP,CCK and their receptors in Spleen Qi deficiency rats.2.Electroacupuncture at “Tsusanli” acupoint participating in the regulation of mitochondrial energy metabolism and improve Spleen Qi deficiency syndrome by regulating the abnormal expression of NRF1 and NRF2 genes and proteins in muscle tissue of rats with Spleen Qi deficiency. |
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