| Objective:Lipolysis injection is an effective method for the treatment of local subcutaneous fat accumulation.Its representative drug deoxycholate often leads to adverse reactions,such as pain,swelling,sclerosis and numbness,and even includes nerve damage,dysphagia,vascular events,neutrophil dermatosis,measles and other complex adverse reactions.To a certain extent,the application and popularity of injection is limited.Experimental studies have shown that myricetin and fat metabolism may be related.This study combined network pharmacology and animal experiments to study whether myricetin can reduce subcutaneous local fat and the possible mechanism.Methods:It was hypothesized that myricetin has the effect of affecting lipid metabolism,and the target of myricetin was obtained from the Pubchem database,the gene set of myricetin was constructed,and the PPI network was used for visualization.Compared with the obesity-related gene set and the gene set of fat metabolism pathway,through comprehensive analysis,it can be predicted whether they have correlation.To analyse the gene function and signaling pathway of the target,GO and KEGG were utilized.Thirty-six 5-week-old male SPF c57 mice weighing 16.0±4.0g were purchased and divided into 9 cages by random counting table method.An obesity model was established after 1 week of suitable rearing,cages 1 to 8 were fed with high-fat diet(feed composition:60%lard),and cages 9 were fed with ordinary diet(4 mice).After 8 weeks of nourishment,the high-fat diet group mice’s average body weight was 20%greater than that the regular diet group,which means the modeling of obese mice was completed.Randomly dividing the obese mice in cages 1-8 into 4 groups,each with 8 mice.The normal saline group(NS)was given normal saline while MyR1(MyR1.0mg/mL),MyR3(MyR3.0mg/mL)and MyR5(MyR5.0mg/mL)were injected myricetin at 1.0,3.0,5.0mg/mL into the left groin fat,respectively 0.1 mL normal saline into the right groin fat as self control.While in the normal saline group,inject equal amount of 0.1mL normal saline into both sides of the groin fat.The frequency is 3 times a week and the period is 4 weeks.After the last injection,the weight of each group mice was weighed,and the groin adipose tissue was dissected and weighed.HE staining was used to evaluate the morphology and size of adipose cells,and Image J software was used to measure adipose cell diameter.The triglyceride content in the adipose tissue of the groin was measured to evaluate the lipolysis effect.The expressions of PLIN1,SIRT1 and ATGL in adipose tissue were detected by enzyme-linked immunospecific assay.The measurement data were expressed as mean ± standard deviation,and matched analysis was performed for comparison between control and treatment side.Differences among different groups were analyzed by one-way analysis of variance(ANOVA),and multiple tests were conducted after the variance analysis.Statistical analysis of the data was conducted using SPSS 26.0 software,with P<0.05 indicating a statistically significant difference and P<0.01 indicating an extremely significant one.Results:Network pharmacological analysis:100 gene targets were selected.A PPI visual network map of 100 nodes and 742 edges was constructed through STRING.Using GO and KEGG analysis to show that the target of myricetin was related to protein phosphorylation,hydrolase and protein kinase.KEGG pathway analysis showed that myricetin was related to metabolism.The shared genes accounted for 91%,61%,92%of the myricetin gene set compared with the obesity gene set constructed by GeneCards and DisGeney databases and their combination.The lipid metabolism gene set was constructed through Pathoards,and compared with the myricetin gene set,the shared genes accounted for 16%myricetin gene set,all of which indicated that myricetin was associated with obesity and lipid metabolism.Establishment of obese mouse model:After adaptive feeding the average body weight of high-fat diet group and the regular diet group was at the same leverl(P>0.05).The average body weight of mice in high fat diet group was higher than that of ordinary diet after 5 weeks feeding(P<0.05).At week 8,the average body weight of mice fed with high fat diet was 21%higher than that fed with ordinary diet.An obese mouse model was successfully established.Ingunial fat weight:The fat weight of MyR1,MyR3 and MyR5 groups on the myricetin treated side was 47.0±12.2mg,55.2±12.4mg,68.3±12.8mg(P>0.05),compared with their control side decreased by 25.8%,17.7%and 24.0%respectively(P<0.05),decreased by 46.8%,37.5%and 22.7%respectively in the NS group(P<0.05);The fat weight of the NS group was 88.3±26.3mg and 89.3±19.1mg(P>0.05).The fat weight of the MyR3 and MyR5 groups was 29.0%and 24.6%lower than that of the NS group(P<0.05).Histological changes:The mean diameter of myricetin treated adipocytes in MyR1,MyR3 and MyR5 groups was 75.67±15.24μm,80.16±10.83μm and 89.18±9.57μm respectively(P>0.05).Compared with the control group,they were decreased by 31.2%,29.8%and 21.0%(P<0.05),and compared with the NS group,they were decreased by 33.7%,29.7%and 21.8%(P<0.05).The mean diameters on both sides of the NS group were 114.07±15.38μm and 116.5±23.29μm,respectively(P>0.05).The mean diameter of the control side in the four groups have no significant differences(P>0.05).ELISA:The PLIN1 in the myricetin treated side of MyR1,MyR3 and MyR5 groups was lower than that in the control side(P<0.05),and the PLIN1 in MyR5 group was lower than that in MyR1 and MyR3 groups(P<0.05),and the both sides of PLIN1 in NS group have no significant differences(P<0.05),indicating that the expression level of PLIN1 could be reduced by myricetin treatment,and the expression level of PLIN1 was negatively correlated with the concentration of myricetin after reaching a certain concentration.ATGL expression levels in MyR1,MyR3,MyR5 and NS groups have no significant differences on both sides(P>0.05).SIRT1 expression levels of MyR1,MyR3 and MyR5 groups were higher than those of normal saline group(P<0.05).SIRT1 expression levels of MyR1,MyR3 and MyR5 groups were higher than those of control group(P<0.05),and the expression levels of SIRT1 in NS group have no significant differences on both sides(P>0.05).The expression level of control side of MyR3 and MyR5 group was higher than that of NS group(P<0.05),and the expression level of control side of MyR1 group and NS group have no significant differences(P>0.05).Triglyceride content:The triglyceride content of myricetin treated side in groups MyR3 and MyR5 was respectively 16.76±3.03mg/g and 16.24±0.22mg/g(P>0.05),were decreased by 19.8%and 13.4%(P<0.05)compared with the control side,and were lower than those in MyR1 and NS groups(P<0.05).The contents on both sides of MyR1 and NS groups have no significant differences(P>0.05).The content of the four groups in the control side have no significant differences(P>0.05).Conclusion:Network pharmacology suggests that myricetin is correlated with obesity and lipid metabolism.Intra-adipose injection of myricetin can reduce local fat,and the mechanism may be related to the decreased expression of PLIN1 and the increased expression of SIRT1and ATGL. |
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