| Purpose:To explore the effects of Catgut Embedding at Shenshu,Pishu and Ganshu points on energy metabolism of adipose tissue,and to elaborate the therapeutic mechanism of Catgut Embedding at Shenshu,Pishu and Ganshu points on perimenopausal obesity,so as to provide theoretical basis for clinical prevention and treatment of perimenopausal obesity.Material and method:1.Animal grouping and modeling: 48 SPF-grade female 8-week-old SD rats with a body mass of(220±10)g were provided by Liaoning Changsheng Biotechnology Co.,Ltd.48 female SD rats were divided into 4groups,including blank group,sham-operated group,OVX group and catgut embedding group,12 rats in each group.All animals were feeding in Animal Center of Liaoning University of Traditional Chinese Medicine,the feeding temperature is 20-24℃,humidity is 40%-70%.Rats of OVX group and catgut embedding group were used to establish perimenopausal model by bilateral ovariectomy,while sham-operated group only removed periovarian adipose mass.The whole experimental process conforms to the "Guiding Opinions on Treating Experimental Animals" and the experimental scheme is examined by the Ethics Review Committee of Liaoning University of Traditional Chinese Medicine.2.Intervention methods: Rats recovered 10 days after OVX operation.The catgut embedding group was treated with Catgut Embedding at Shenshu,Pishu and Ganshu points,once every 10 days,for 8 times.The rest of the rats were only bound to the fixator without catgut embedding.During the whole experiment,all the animals ate and drank freely,and the illumination: darkness = 12h: 12 h.The feed was SPF grade rat feed.3.Indicator detection:3.1Experiment 1: Record the changes of food intake and body weight of rats in each group during the experiment.At the end of the experiment,the rats were executed on an empty stomach for 12 hours.After anesthesia,the body length and abdominal circumference of the rats were measured accurately.Lee’s index was calculated.WAT of uterus,inguinal subcutaneous,perinephric and BAT of scapular region was peeled.Uterine index and WAT and BAT weight percentage were calculated.Blood was collected from abdominal aorta and blood lipid was detected by automatic biochemical analyzer.The serum estradiol,ovarian stimulating hormone,luteinizing hormone,leptin and adiponectin were detected by Elisa method.3.2Experiment 2: Extraction of BAT from Rats,and tissues used for molecular biology detection were quickly put into liquid nitrogen,and tissues used for histological staining were fixed in polyformaldehyde.Observe the morphology and structure of BAT of rats in each group by HE staining;detect the expression of UCP1 protein in BAT of rats in each group by immunohistochemical staining;measure the content of cAMP in BAT of rats in each group by Elisa method;detect the expression of PKA,CREB,PPARγ,PGC1,UCP1 mRNA in BAT rats in each group by qRT-PCR method;The expressions of PKA,p-CREB/CREB,PPARγ,PGC1 and UCP1 in BAT of rats in each group were determined by Western Blotting method.3.3Experiment 3: Extraction of WAT from Rats,and tissues used for molecular biology detection were quickly put into liquid nitrogen,and tissues used for histological staining were fixed in polyformaldehyde.Observe the morphology and structure of WAT of rats in each group by HE staining;detect the expression of UCP1 protein in WAT of rats in each group by immunohistochemical staining;measure the content of cAMP in WAT of rats in each group by Elisa method;detect the expression of PKA,CREB,PPARγ,PGC1,UCP1 mRNA in WAT rats in each group by qRT-PCR method;The expressions of PKA,p-CREB/CREB,PPARγ,PGC1 and UCP1 in WAT of rats in each group were determined by Western Blotting method.4.Statistical analysis: All statistical data were analyzed and processed by SPSS 18.0 statistical software.Measurements were expressed as?X±s.Independent sample t test was used to compare the data between statistical groups,and P < 0.05 showed significant difference.Results:1.Experiment 1:1.1The results of uterine weight and Elisa test of serum E2,FSH and LH levels in rats of each group showed that: compared with sham-operated group,the weight of uterus and the level of serum reproductive hormone in OVX group decreased,the wet weight of uterus and uterine index decreased,the level of serum E2 decreased,the level of FSH and LH increased,the difference was statistically significant(P < 0.05,P < 0.001).Compared with OVX group,the wet weight of uterus and uterine index,the level of serum E2 of catgut embedding group increased,the level of FSH and LH were decreased,the difference was statistically significant(P < 0.05,P < 0.001).There were no significant differences between the blank group and the sham-operated group(P > 0.05).1.2The results of obesity-related indexes showed that: compared with sham-operated group,OVX group rats ate more,their body weight and Lee’s index increased,their abdominal circumference increased,the percentage of fat weight and white fat weight increased(P < 0.05,P < 0.001).Four items of blood lipids showed that the levels of TC,TG and LDL in serum of OVX rats increased,while the levels of HDL decreased(P < 0.05,P < 0.001).Compared with OVX group,food intake of catgut embedding group decreased,body weight and Lee’s index decreased,the abdominal circumference decreased,the WAT weight percentage decreased,the difference was statistically significant(P < 0.05,P < 0.001).The four items of blood lipid showed that the levels of serum TG,LDL decreased and HDL increased in catgut embedding group,the difference was statistically significant(P < 0.05,P < 0.001).There were no significant differences between the blank group and the sham-operated group(P > 0.05).1.3The results of Elisa test of serum adipokine content in rats of each group showed that: compared with sham-operated group,the serum leptin level in OVX group increased(P < 0.001),the adiponectin level decreased(P < 0.001).Compared with OVX group,the serum leptin level in catgut embedding group decreased,the difference was statistically significant(P < 0.001),and the adiponectin level increased,the difference was statistically significant(P < 0.001).There were no significant differences between the blank group and the sham-operated group(P > 0.05).2.Experiment 2:2.1The results of HE staining and UCP1 immunohistochemical staining of BAT of each group showed that: compared with sham-operated group,brown adipocytes in BAT of OVX group were disorderly arranged and enlarged in volume.The results of immunohistochemical staining showed that UCP1 positive staining was significantly reduced,the difference was statistically significant(P < 0.001).Compared with OVX group,adipocytes in catgut embedding group were smaller,and the positive rate of UCP1 immunohistochemical staining was higher than that in sham-operated group,the difference was statistically significant(P < 0.001).There were no significant differences between the blank group and the sham-operated group(P > 0.05).2.2The results of Elisa test of cAMP content in BAT of each group showed that: compared with sham-operated group,the content of cAMP in BAT in OVX group decreased significantly(P < 0.001).Compared with OVX group,the content of cAMP in BAT in catgut embedding group increased,the difference was statistically significant(P < 0.001).There were no significant differences between the blank group and the sham-operated group(P > 0.05).2.3The results of qRT-PCR test of expression of PKA、CREB、PPARγ、PGC1、UCP1 mRNA in BAT of each group showed that: compared with sham-operated group,expression of PKA、CREB、PPAR、PGC1、UCP1 mRNA in BAT of OVX group decreased,the difference was statistically significant(p<0.05,p<0.001).Compared with OVX group,expression of PKA、CREB、PPAR、PGC1、UCP1 mRNA in BAT of catgut embedding group increased,the difference was statistically significant(p<0.05).There were no significant differences between the blank group and the sham-operated group(P > 0.05).2.4The results of Western blotting test of protein expression of PKA、p-CREB /CREB 、PPARγ、PGC1、UCP1 in BAT of each group showed that: compared with sham-operated group,protein expression of PKA、PPARγ、PGC1、UCP1 in BAT of OVX group decreased,and the ratio of p-CREB/CREB decreased,the difference was statistically significant(P < 0.05,P < 0.001).Compared with OVX group,the expression of PKA,PPARγ,PGC1,UCP1 protein and the ratio of p-CREB/CREB in BAT of catgut embedding group increased,the difference was statistically significant(P < 0.05,P < 0.001).There were no significant differences between the blank group and the sham-operated group(P > 0.05).3.Experiment 3:3.1The results of HE staining and UCP1 immunohistochemical staining of WAT of each group showed that: the size,shape and arrangement of adipose tissue were normal in the blank group and the sham-operated group,and immunohistochemical staining showed that UCP1 was inherently less expressed,and there was no significant difference between the two groups(P > 0.05).Compared with the sham-operated group,the white adipocytes in the WAT of the OVX group were disordered and enlarged,the difference was statistically significant(P < 0.001),and immunohistochemical staining showed that the positive rate of UCP1 immunohistochemical staining in catgut embedding group was higher than that in OVX group,the difference was statistically significant(P < 0.001).3.2The results of Elisa test of cAMP content in WAT of each group showed that: compared with sham-operated group: the content of cAMP in WAT in OVX group decreased significantly(P < 0.001).Compared with OVX group,the content of cAMP in WAT in catgut embedding group increased,the difference was statistically significant(P < 0.001).There were no significant differences between the blank group and the sham-operated group(P > 0.05).3.3The results of qRT-PCR test of expression of PKA、CREB、PPARγ、PGC1、UCP1 mRNA in WAT of each group showed that: compared with sham-operated group,expression of PKA、CREB、PPAR、PGC1、UCP1 mRNA in WAT of OVX group decreased,the difference was statistically significant(P<0.05).Compared with OVX group,expression of PKA、CREB、PPAR、PGC1、UCP1 mRNA in WAT of catgut embedding group increased,the difference was statistically significant(p<0.05,p<0.001).There were no significant differences between the blank group and the sham-operated group(P > 0.05).3.4The results of Western blotting test of protein expression of PKA、p-CREB /CREB 、PPARγ、PGC1、UCP1 in WAT of each group showed that: compared with sham-operated group,protein expression of PKA、PPARγ、PGC1、UCP1 in WAT of OVX group decreased,and the ratio of p-CREB/CREB decreased,the difference was statistically significant(P < 0.05).Compared with OVX group,the expression of PKA,PPARγ,PGC1,UCP1 protein and the ratio of p-CREB/CREB in WAT of catgut embedding group increased,the difference was statistically significant(P < 0.05,P < 0.001).There were no significant differences between the blank group and the sham-operated group(P > 0.05).Conclusion:1.Embedding catgut at Shenshu,Pishu and Ganshu points can alleviate the sudden change of reproductive hormone in perimenopausal period to some extent and improve the related indexes of obesity;2.Embedding catgut at Shenshu,Pishu and Ganshu points can improve reproductive hormone level,activate the cAMP/PKA signaling pathway in BAT and WAT,thus promote downstream CREB phosphorylation,increase the expression of PPARgamma and PGC1,thereby increase the content of UCP1 in tissues.In this way to increase the energy metabolism of BAT and promote the browning of WAT,thus strengthen the energy metabolism of adipose tissue and play a role in preventing and treating perimenopausal obesity. |
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