| Objective:1 The "vertical abdomen and plumpness" of "cream people" in Neijing is similar to the abdominal obesity of patients with metabolic syndrome(MS),which is characterized by abdominal bulge and sag.This clinical study aims to explore a new method for quantitative evaluation of abdominal obesity and its application value based on the "vertical abdomen and plumpness" theory.2 White adipose tissue(WAT)is induced into beige fat with the characteristics of heat production and energy consumption,that is,WAT browning,which is an effective way to improve obesity.Abdominal obesity belongs to "fat fullness" in traditional Chinese medicine,and "moderate fullness and internal heat" is the basic pathogenesis.Dai-Zong-Fang(DZF)formulated by the tutor with the method of "eliminating phlegm and clearing heat" has a certain curative effect.The purpose of this study is to explore the effect and mechanism of DZF on glucose and lipid metabolism and WAT browning by using the obese C57BL/6J mouse model and the mature 3T3-L1 adipocytes as research carriers.Methods:1 Clinical study:150 cases were randomly collected and divided into 100 cases in MS group and 50 cases in normal group.The age and medical history of the subjects were collected,the height,weight,waist circumference,and hip circumference were measured,and the BMI,WHR,and WHtR were calculated.Set and manually measure the evaluation indexes of abdominal protrusion(abdominal protrusion,relative abdominal protrusion,abdominal protrusion/height,relative abdominal protrusion/height)and abdominal ptosis(abdominal height,relative abdominal height,abdominal height/height,relative abdominal height/height).The distribution of body fat was detected by a human body composition analyzer.The differences in abdominal protrusion and ptosis between the two groups were analyzed by independent t-test.Partial correlation analysis was used to analyze the relationship between abdominal protrusion and ptosis and visceral fat in MS group.The relationship between abdominal protrusion and ptosis and TCM syndromes in MS group was analyzed by independent t-test.2 In vivo experiment:the obesity model of C57BL/6J mice was established by diet induction,and intervened with high and low doses of DZF and metformin(positive control drug)for 6 weeks.After anesthesia,the mice were weighed,their body length and abdominal circumference were measured,and the Lee’s index was calculated.After the mice were killed,the serum FBG,TG,TC,HDL-C and LDL-C were detected by automatic biochemical instrument.The level of serum NEFA was detected by a colorimetric assay-kit.White adipose tissue and brown adipose tissue in scapula were taken from perirenal,epididymis and groin.The wet weight of fat was weighed,and the white and brown fat coefficients were calculated.The size and browning of adipocytes were observed by HE staining.The browning markers UCP-1 and PGC-1α mRNA and the mRNA of the key molecules of the camp-PKA pathway were detected by PCR.3 In vitro experiment:the white adipocyte model 3T3-L1 preadipocytes were induced to differentiate and mature.The cytotoxicity of DZF was detected by the CCK8 method.The intervention concentrations of DZF were 0.4 mg/mL(low concentration group)and 0.8 mg/mL(high concentration group).After 48 hours of intervention,the morphological changes of adipocytes were detected by double fluorescence staining of BODIPY and DAPI,the changes of intracellular TG content were measured by GPO-PAP method,and the expression of the lipid synthesis protein SREBP-1,browning protein UCP-1 and PGC-1α were detected by Western blotting.The number of mitochondria was measured by Mito-tracker Green fluorescence staining.The UCP-1,PGC-lα and PRDM16 mRNA were detected by PCR.Western bloting was used to detect the expression of PKA and AMPK,the key proteins in browning pathway,and the expression changes of related proteins were detected after adding AMPK inhibitor dorsomorphin and PKA inhibitor H-89 dihydrochloride.Results:1 Clinical research:(1)Abdominal protrusion and ptosis of MS were compared with normal subjects:①Abdominal protrusion:In male MS group,abdominal protrusion and relative abdominal protrusion were 5.64 cm and 4.9 cm higher than those in normal group respectively(P<0.05),abdominal protrusion/height and relative abdominal protrusion/height were 0.03 higher than those in normal group(P<0.05).In female MS group,abdominal protrusion and relative abdominal protrusion were 6.14 cm and 4.31 cm higher than those in normal group(P<0.05),abdominal protrusion/height and relative abdominal protrusion/height were 0.04 and 0.03 higher than those in normal group(P<0.05).② Abdominal ptosis:In male MS group,abdominal height and relative abdominal height were 3.16 cm and 2.37 cm higher than those in normal group(P<0.05),abdominal height/height and relative abdominal height/height were 0.01 higher than those in normal group(P<0.05).In female MS group,abdominal height and relative abdominal height were 1.67 cm(P<0.05)and 1.8 cm lower than those in normal group(P>0.05),abdominal height/height and relative abdominal height/height were 0.01 than those in normal group(P<0.05).(2)The relationship between abdominal protrusion and ptosis of MS and visceral fat:①Abdominal protrusion:excluding the factors of age,weight,and height,there were positive correlations between abdominal protrusion and visceral fat volume,visceral fat percentage,visceral fat area and visceral fat level in male MS patients(r>0,P<0.05).There was no correlation between abdominal protrusion and visceral fat index in MS women.② Abdominal ptosis:Excluding age,weight,and height,there was no correlation between abdominal ptosis and visceral fat in male MS patients.Abdominal height,abdominal height/height were positively correlated with visceral fat rate in female MS patients(r>0,P<0.05).(3)The relationship between abdominal protrusion and ptosis of MS and TCM syndromes:① Abdominal protrusion and relative abdominal protrusion of MS in the phlegm-heat syndrome group were 6.20%and 8.28%higher than those in the non-phlegm-heat syndrome group,respectively(P<0.05).② Abdominal ptosis:abdominal height,abdominal height/height,and relative abdominal height were increased by 6.65%,1.67%,and 10.65%in the phlegm-heat syndrome group compared with those in the non-phlegm-heat syndrome group(P<0.05).The abdominal height,abdominal height/height,relative abdominal height,and relative abdominal height/height of the spleen deficiency dampness syndrome group were 6.65%,1.67%,and 10.65%lower than those of the non-spleen deficiency dampness syndrome group(P<0.05),respectively.2 In vivo experiment:(1)Effects of DZF on glucolipid metabolism in the C57BL/6J obese mice:① Compared with the model group,FBG was decreased by 21.6%in the DZF-H group(P<0.01),was decreased by 18.8%in the DZF-L group(P<0.05)and was decreased by 23.6%in the metformin group(P<0.001).②Compared with the model group,TC,TG and LDL-C in the DZF-H group were decreased by 32.0%(P<0.001),31.3%(P<0.001)and 15.9%(P<0.01),respectively;TC,TG and LDL-C in the DZF-L group were decreased by 8.1%,24.8%(P<0.05)and 8.5%,respectively.TC,TG and LDL-C were decreased by 22.2%(P<0.001),14.3%and 12.9%(P<0.05)in the metformin group,respectively.NEFA in each administration group was lower than that in the model group,but there was no statistical difference(P>0.05).(2)The effect of DZF on the browning of white fat in the C57BL/6J obese mice:①Compared with the model group,eWAT,iWAT,pWAT and BAT in the DZF-H group were decreased by 49.2%(P<0.001),48.2%(P<0.05),62.8%(P<0.001)and 30.5%,respectively.EWAT,iWAT,pWAT and BAT were decreased by 50.25%(P<0.001),32.3%(P<0.05),52.4%(P<0.001)and 21.3%in the metformin group,respectively.DZF-L intervention did not significantly reduce the fat mass in all parts(P>0.05).② Compared with the model group,the HE staining of iWAT in the DZF-H group showed that the volume of adipocytes decreased,and the number of mitochondria increased with clear structure under electron microscope.③ Compared with the model group,UCP-1 and PGC-1α mRNA in eWAT and iWAT in the DZF-L/H group and the metformin group were increased to varying degrees(P<0.05 or P<0.01).④Compared with the model group,cAMP and PKA in iWAT in the DZF-H group and the metformin group were significantly increased(P<0.05 or P<0.01 or P<0.001).3 In vitro experiment:(1)The effects of DZF on the browning of 3T3-L1 adipocytes:① Compared with the con group,BODIPY493/503 staining showed that the morphology of adipocytes was smaller and the number of lipid droplets was relatively increased in the high and low concentration DZF groups.② Compared with the con group,Mito-Tracker Green staining showed that the number of mitochondria in adipocytes in the DZF high and low concentration groups was significantly increased.③Compared with the con group,the expression of UCP-1 and PGC-1α in the high concentration DZF group were significantly increased(P<0.01).The expression of PGC-1α was significantly increased in the low concentration DZF group(P<0.01).and the expression of UCP-1 had an increasing trend.(2)The effects of DZF on the PKA/AMPK pathway:① Compared with the con group,the expression of PKA and pCREB in the DZF groups with different concentrations were significantly increased(P<0.01 or P<0.05),while the expression of CREB was not significantly changed.Compared with the DZF group,the expressions of UCP-1 and PGC-1αin the DZF+H-89 dihydrochloride group were significantly decreased(P<0.05).②Compared with the con group,the expression of pAMPK in the DZF group was significantly increased(P<0.01),while the expression of AMPK was not significantly changed.Compared with the DZF group,the expressions of UCP-1 and PGC-1α in the DZF+Dorsomorphin group were significantly decreased(P<0.01).Conclusions:1 Clinical research:(1)The degree of abdominal protrusion in MS patients was higher than in the normal population in both males and females.The degree of abdominal ptosis in male MS patients was lower than that in the normal population(abdominal lifting was obvious),and the degree of abdominal ptosis in female MS patients was higher than that in the normal population(abdominal ptosis was obvious).(2)The evaluation of abdominal protrusion may be able to evaluate the visceral fat of male MS patients to a certain extent.The evaluation of abdominal sagging may be able to evaluate the visceral fat of female MS patients to a certain extent.(3)The increase of abdominal protrusion and abdominal lifting in MS patients may be related to the phlegm-heat syndrome,and abdominal ptosis may be related to the pleen deficiency dampness syndrome.2 In vivo experiment:(1)DZF can reduce body weight,abdominal circumference,Lee’s index,FBG and blood lipid of the obese C57BL/6J mice,reduce obesity and improve glucolipid metabolism disorder.(2)DZF decreased eWAT,iWAT,pWAT and WAT coefficient in the obese C57BL/6J mice,promoted the expression of UCP-1 and PGC-1α,and mitochondrial remodeling and induced the Browning of eWAT and iWAT.3 In vitro experiment:(1)DZF can reduce TG content in the 3T3-L1 adipocytes,inhibit lipid accumulation,decrease the expression of SREBP-1 and inhibit adipogenesis.(2)DZF can increase the number of mitochondria in the 3T3-L1 adipocytes,promote the expression of UCP-1,PGC-1α and PRDM16,and induce browning reaction.Its mechanism is related to the activation of PKA/AMPK signaling pathway. |
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