| Purpose:In clinical practice,it is found that there are more T2DM patients with abdominal obesity,among which a considerable proportion of patients are identified as having spleen deficiency and dampness,and the regular treatment of these patients with the traditional Chinese medicine compound Yitangkang is effective,but the specific mechanism has not been fully explored.In order to clarify the scientificity,practicality and importance of the clinical"spleen-based treatment"of abdominal obesity in T2DM and to prove the mechanism of the Chinese medicine compound Yitangkang’s intervention in abdominal obesity in T2DM,the following three parts of the study were set up.In the clinical research part,the distribution of evidence elements in T2DM abdominal obesity patients and the influence of the main evidence elements on clinical indexes were analyzed by the method of evidence element analysis;in the animal experiment part,whether the Chinese medicine compound Yitangkang has an intervention effect on white fat browning in db/db mice was investigated,and whether the mechanism is related to the activation of AMPK.In the cell experiment,the effect of the herbal compound Yitangkang containing serum on the expression of browning signature genes in mature adipocytes induced by 3T3-L1 cells was investigated to further clarify whether the mechanism is related to the regulation of AMPK/PGC-1αsignaling pathway,which in turn regulates mitochondrial biogenesis and promotes white fat browning.Material and method:Article 1:Distribution and clinical study of T2DM abdominal obesity patients with TCM evidence elementsWe collected patients with T2DM abdominal obesity who attended the endocrinology department and physical examination center of the Affiliated Hospital of Liaoning University of Chinese Medicine from September 2021 to December 2022,and designed the"Patient Evidence Information Collection Form"for T2DM abdominal obesity used in this study according to the research design method of Professor Zhu Wenfeng’s"Evidence Element Identification",combined with expert questionnaires."The data were analyzed using statistical methods to explore the distribution pattern of T2DM abdominal obesity patients’TCM evidence elements and to conduct statistical analysis on the clinical data of key disease locations and disease evidence elements.Article 2:Effect of Chinese medicine compound Yitangkang on white fat browning in db/db miceForty-five SPF-grade 5-week-old male C57BL/Ks J db/db mice were selected and randomly divided into model group,liraglutide group,Yitangkang low-dose group,Yitangkang medium-dose group and Yitangkang high-dose group,with 9 mice in each group;9 male SPF-grade 5-week-old mice in the same cage were also selected as the experimental blank group.From the 10th week age,mice in the low,medium and high dose groups were gavaged with 15g·kg-1·d-1、30g·kg-1·d-1、60g·kg-1·d-1 of Yitangkang decoction for 4 weeks,and 200μg·kg-1 liraglutide was injected subcutaneously daily for 4 weeks in the liraglutide group,and distilled water was given to the blank group and the model group for 4 weeks.The daily dose of liraglutide by gavage was based on the daily adult dose combined with the equivalent dose by body surface area between human and mice.The morphological changes of white adipose in each group were observed by HE staining;the number and morphological changes of mitochondria in white adipose cells in each group were observed by transmission electron microscopy;the expression levels of browning-specific proteins such as UCP-1,PRDM16,PPAR-γand PGC-1αin white adipose tissue of mice were detected by immunohistochemistry;the expression levels of UCP-1,PRDM16,PPAR-γ,PGC-1α,and p-AMPK,AMPK proteins in white adipose tissue of mice were detected by Western blot.Article 3:Study on the browning mechanism of adipocytes induced differentiation of3T3-L1 cells by Chinese herbal compound YitangkangTwenty SPF-grade SD male rats were selected for the preparation of low,medium and high doses of herbal compound Yitangkang-containing serum and blank serum.After the gavage cycle,the rats were anesthetized with 3%pentobarbital sodium intraperitoneally,and the blood was collected from the abdominal aorta to prepare the drug-containing serum and the blank serum.The 3T3-L1 preadipocytes were induced to differentiate into mature adipocytes according to the traditional"cocktail"induction method.The optimal dose of CCK-8 method was used to screen the dose of the drug-containing serum for the induction of differentiation of mature adipocytes from 3T3-L1 preadipocytes.The differentiated mature adipocytes were divided into the blank serum group,the herbal compound Yitangkang group,and the herbal compound Yitangkang+AMPK inhibitor group,and the intervention was performed according to the established design for 48 h.After the end of intervention,the cells in each group were collected and the effect of AMPK,p-AMPK,PGC-1α,NRF1,NRF2,TFAM,PRDM16,PPAR-γand UCP-1 protein expression levels in each group of adipocytes were detected using Western blot method.Results:Article 1:Distribution of TCM evidence and clinical study of abdominal obese patients with T2DM1.A total of 340 patients with T2DM abdominal obesity were included in this study,including 190 male patients（55.88%）,age（49.86±10.03）,maximum age 60 years and minimum age 23 years;150 female patients（44.12%）,age（52.63±9.21）,maximum age 60years and minimum age 18 years.2.340 patients with abdominal obesity in T2DM were mainly focused on the spleen（148cases,43.53%）,followed by the liver（111 cases,32.65%）,kidney（87 cases,25.59%）,stomach（70 cases,20.59%）,gallbladder（40 cases,11.76%）,meridians（40 cases,11.76%）,lung（27 cases,7.94%）,and heart（7.94%）.7.94%),heart（16 cases,4.71%）;the results of the pathogenic evidence showed that the actual pathogenic evidence was mainly concentrated in dampness（152 cases,44.71%）,followed by Qi stagnation（133 cases,39.11%）,blood stasis（119 cases,35.00%）,drinking（77 cases,22.65%）,phlegm（50 cases,14.71%）,heat（48 cases,14.12%）,and water stagnation（14.12%）.14.12%),water retention（43 cases,12.65%）,and food accumulation（27 cases,7.94%）;deficiency pathological elements were mainly distributed in Qi deficiency（156 cases,45.88%）,followed by Yin deficiency（130 cases,38.24%）,Yang deficiency（86 cases,25.29%）,Blood deficiency（61 cases,17.94%）,and Qi trapping（43 cases,12.65%）.3.According to the results of the distribution of the main pathological evidence elements and pathological evidence elements,the subjects were labeled as spleen group and non-spleen group according to whether they contained the pathological evidence element spleen;the subjects were labeled as wet group and non-damp group according to whether they contained the actual pathological evidence element damp;and the subjects were labeled as Qi deficiency group and non-Qi deficiency group according to whether they contained the deficiency pathological evidence element Qi deficiency.The results showed that the BMI,WC,and WHR of patients in the spleen group were higher than those in the non-spleen group compared with those in the non-spleen group,P<0.05,and the differences were statistically significant;the FPG,Hb A1c,TG,TC,and LDL-C of patients in the spleen group were higher than those in the non-spleen group,and the HDL-C of patients in the spleen group was lower than that in the non-spleen group,P<0.05,and the differences were statistically significant;compared with those in the non-wet group,BMI,WC,and WHR of patients in the wet group were higher than those in the non-wet group,P<0.05,and the difference was statistically significant;compared with patients in the non-wet group,FPG,Hb A1c,TG,and TC of patients in the wet group were higher than those in the non-wet group,P<0.05,and the difference was statistically significant;HDL-C and LDL-C of patients in the wet group compared with the non-wet group,P>0.05,the difference is not statistically significant;compared with the non-Qi deficiency group,the BMI and WC of patients in the Qi deficiency group were higher than those in the non-Qi deficiency group,P<0.05,and the difference was statistically significant;the WHR of patients in the Qi deficiency group compared with those in the non-Qi deficiency group,P>0.05,and the difference was not statistically significant;compared with patients in the non-Qi deficiency group,HDL-C of patients in the Qi deficiency group was lower than that of the non-Qi deficiency group,P<0.05,and the difference was statistically significant;FPG,Hb A1c,TG,TC,and LDL-C of patients in the Qi deficiency group were compared with those in the non-Qi deficiency group,P>0.05,and the difference was not statistically significant.Article 2:Effects of Chinese herbal compound Yitangkang on white fat browning in db/db mice1.Body mass of mice in each group;before treatment,the body mass of mice in the blank group was significantly lower than that of all other groups,P<0.05,and no significant difference was seen between the remaining groups,P>0.05;after treatment,no significant difference was seen in the body mass of mice in the low dose group of Chinese medicine compared with that of mice in the model group,P>0.05,while the body mass of mice in the middle and high dose groups of Chinese medicine and liraglutide group was significantly different compared with that of the model group,P<0.05;the body mass of mice in the three groups of Chinese medicine medium and high dose group and liraglutide group did not show significant difference after treatment,P>0.05.2.Before treatment,the fasting blood glucose level of mice in the blank group was significantly lower than that of other groups,P<0.05,while no significant difference was found between the remaining groups,P>0.05.After treatment,compared with the model group,no significant difference was found in the fasting blood glucose level of mice in the low dose group of Chinese medicine,P>0.05,while the fasting blood glucose level of mice in the middle and high dose groups of Chinese medicine and liraglutide group was significantly different from that of the model group,P>0.05.Compared with the model group,the difference was significant,P<0.05;after treatment,the fasting blood glucose levels of mice in the middle and high dose groups of Chinese medicine and liraglutide group were not statistically different,P>0.05.The lipid levels of mice in each group after drug treatment:TG,TC and LDL-C of mice in the model group were significantly higher than those in the blank group,and HDL-C was significantly smaller than that in the blank group,P<0.05;compared with the model group,TG,TC and LDL-C of mice in the Chinese medicine medium and high dose groups were significantly improved,and HDL-C was significantly higher,P<0.05;the effects of Chinese medicine medium dose and Chinese medicine high dose were comparable,P>0.05.3.HE staining of white adipose tissue of mice in each group:white adipose tissue of blank group could be observed that white adipocytes were closely arranged,the cells were mostly round-like in shape under the microscope,with different sizes,and the cell volume was moderate in general;the cell volume of model group was obviously increased,the arrangement was crowded and dense,and some cells were extruded and deformed from each other;the adipocytes in low-dose,medium-dose,high-dose and liraglutide groups of Chinese medicine were significantly smaller than those in model group,and the volume of adipocytes was significantly smaller than that in model group.The cell volume of the three different dose groups of Chinese medicine showed a decreasing trend with the increase of dose,and the cell volume of the middle and high dose groups of Chinese medicine was comparable to that of liraglutide group.4.The number and morphology of mitochondria of white adipocytes in each group were observed by transmission electron microscopy:the mitochondria of white adipocytes in the blank group were elliptical in shape,and the number was less,and the mitochondrial shape was intact;compared with the blank group,the mitochondria of white adipocytes in the model group were less in number and showed vacuole-like changes,and the mitochondrial cristae were broken or even disappeared under the microscope;compared with the model group,the mitochondria in the low,medium and high dose groups of traditional Chinese medicine and the liraglutide group Compared with the model group,the number of mitochondria in white adipocytes was increased in the low,medium and high dose groups and the liraglutide group.5.The expression levels of UCP-1,PRDM16,PPAR-γand PGC-1αproteins in white adipose tissue of mice were observed using immunohistochemistry.The results showed that the expression levels of UCP-1,PRDM16,PPAR-γand PGC-1αin white adipose tissue of mice in the db/db group decreased compared with the db/m group（P<0.05）;the expression levels of the above proteins in all the drug intervention groups increased compared with the db/db group（P<0.05）;among the four drug intervention groups,the expression levels of the above proteins were higher in the middle and high dose groups of Yitangkang and the liraglutide group than in the low dose group of Yitangkang（P<0.05）.6.The expression levels of UCP-1,PRDM16,PPAR-γ,PGC-1α,AMPK and p-AMPK proteins in white adipose tissue of mice were detected by Western Blot method.The results showed that the expression levels of UCP-1,PRDM16,PPAR-γ,PGC-1αand p-AMPK/AMPK in white adipose tissue of mice in the db/db group decreased compared with the db/m group（P<0.05）;the expression levels of the above proteins and p-AMPK/AMPK values in all drug intervention groups increased compared with the db/db group（P<0.05）;among the four drug intervention groups,the above protein expression levels and p-AMPK/AMPK values were higher in the middle and high dose groups of Yitangkang and liraglutide than in the low dose group of Yitangkang（P<0.05）;there was no significant difference between the middle and high dose groups of Yitangkang and liraglutide（P>0.05）.Article 3:Study on the mechanism of adipocyte browning in 3T3-L1 cells induced differentiation by Chinese herbal compound Yitangkang1.Oil red O staining observed that the mature adipocytes with successful induced differentiation were larger in size and rounder in morphology than the undifferentiated precursor adipocytes,and most of the cells in the field of view could be seen to contain high bright red-brown lipid droplets,some of which had fused from dispersed small lipid droplets into larger lipid droplets.2.CCK-8 results showed that no significant difference was seen in cell viability between the groups of high,medium and low dose groups of herbal drug-containing serum.For the screening of the optimal concentration of the subsequent cell intervention culture,considering that since the medium dose of TCM is the conventional dose for clinical treatment and the in vivo experiment in paper II also showed that the effect of the medium dose of Yitangkang on white fat browning in db/db mice was not significantly different from the effect of the high dose of TCM,the medium dose was used as the intervention dose of the drug in all subsequent experiments.3.The Western Blot results showed that compared with the blank serum group,the p-AMPK/AMPK values of adipocytes in the Yitangkang-containing serum group were significantly up-regulated,and the expression levels of PGC-1αand NRF1,NRF2,TFAM mitochondrial biogenesis-related proteins were increased,and the expression of PRDM16,PPAR-γand UCP-1,the signature proteins related to white fat browning,were also increased（P<0.05）.In contrast to the Yitangkang-containing serum group,the p-AMPK/AMPK values of the Yitangkang-containing serum+AMPK inhibitor group decreased,the expression levels of PGC-1αand NRF1,NRF2 and TFAM mitochondrial biogenesis-related proteins were down-regulated,and the expression levels of white fat browning-related signature proteins PRDM16,PPAR-γand UCP-1 were down-regulated,and the differences were statistically significant（P<0.05）.Conclusion:1.The distribution of TCM evidence elements in abdominal obese patients with T2DM,the main pathological evidence elements are:spleen,liver,kidney,etc.;the main real pathological evidence elements are:dampness,Qi stagnation,blood stasis,etc.;the main deficiency pathological evidence elements are:Qi deficiency,Yin deficiency,Yang deficiency,blood deficiency,Qi trapping.The main pathogenic factor,spleen,had effects on the levels of BMI,WC,WHR,FPG,Hb A1c,TG,TC,HDL-C,LDL-C,etc.;the main actual pathogenic factor,damp,had effects on the levels of BMI,WC,WHR,FPG,Hb A1c,TG,TC;the main deficiency pathogenic factor,Qi deficiency,had effects on the levels of BMI,WC,HDL-C.2.The Chinese medicine compound Yitangkang can improve the disorder of glucolipid metabolism in db/db mice,it also can improve fasting blood sugar and TG,TC,HDL-C,LDL-C levels,control the growth of body mass,promote the browning of white adipose tissue in db/db mice,upregulate the browning-related signature proteins such as UCP-1,PRDM16,PPAR-γ,PGC-1αin white adipose tissue,increased the number of mitochondria in adipocytes and improved mitochondrial morphology.Meanwhile,the herbal compound Yitangkang promoted the process of white fat browning,which may be related to the activation of AMPK.3.The mechanism of promoting browning of mature adipocytes from 3T3-L1-induced differentiation may be related to the regulation of AMPK/PGC-1αsignaling pathway-mediated mitochondrial biogenesis. |
