Preventive Effect And Mechanism Research Of Forsythin Extract In Forsythia Leaves On Obesity Of Rats Induced By High-fat Diet

Objective:The orthogonal test method was used to study the optimal extraction technology of Forsythin in Forsythia leaves,and to explore the preventive effect of Forsythin extract in Forsythia leaves on obesity induced by high-fat diet in rats.Adenosine monophosphate-activated protein kinase(AMPK)/acetyl CoA carboxylase(ACC)/sterol regulatory element binding protein-1c(SREBP-lc)signaling pathway was examined as a target to preliminarily explore the possible molecular mechanisms.At the same time,the influences of Forsythin extract on the structure of intestinal flora and the content of short-chain fatty acids(SCFAs)in obese rats were also studied,which provided scientific basis and theoretical support for the development and utilization of functional food from Forsythia leaf.Methods:(1)Firstly,preparing the Forsythin extract from Forsythia leaves.After crushing the Forsythia leaves,to take an appropriate amount and add 20 times the volume of water,and extraction was performed 2 times at 90℃ for 60 min each time.The filtrate was collected twice.After centrifugation,the supernatant was taken out.Then it was concentrated and freeze-dried into powder by rotary evaporator for subsequent animal experiments.(2)65 male SD rats weighing(200±10)g were adaptively fed for 1 week and then randomly divided into a blank control group(8)and an obesity model group(57)according to their body weight.The blank control group was fed with basic diet,and the obesity model group was fed with high-fat diet.After feeding for 2 weeks,the 57 rats in the obesity model group were ranked by weight gain,and 17 obese-resistant rats with lower body weight gain were eliminated.The 40 obesity-sensitive rats selected were randomly divided into 5 groups according to their body weight.They were model control group,Forsythin extract low,medium and high dose groups and standard group.They were given Forsythin extract 0.2 g/(Kg d),0.4 g/(kg d),1.2 g/(kg d)and standard Forsythin(the mass fraction of Forsythin is 98%)54.3 mg/(kg·d)respectively.The blank control group was given basal feed,and the other groups were given high-fat feed.The blank control group and the model control group were given the same amount of distilled water for 6 weeks of continuous intervention.At the end of the experiment,the body weight was weighed,the body length was measured.After the rats were killed,the serum was separated,the liver,kidney,testis,perirenal fat and peritestinal fat were dissected.The liver and peritesticular fat were taken for hematoxylin-eosin staining.The serum,feces and other tissues were stored in an ultra-low temperature refrigerator at-80℃.Using an automatic biochemical analyzer to detect serum total cholesterol(TC),serum triglyceride(TG),low density lipoprotein cholesterol(LDL-C)and high density lipoprotein cholesterol(HDL-C).Using ELISA method to detect total bile acid(TBA),leptin(LP),free fatty acid(FFA).Western Blot(WB)was used to detect the key protein including the adenosine monophosphate-activated protein kinase(AMPK),acetyl CoA Carboxylase(ACC),sterol regulatory element binding protein lc(SREBP1c),fatty Acid Synthase(FAS)expression levels.Using 16S rDNA high-throughput sequencing technology to analyze the changes of intestinal flora.Using GC-MS method to detect the content of short-chain fatty acids in feces.Results:(1)Optimization of the extraction process of Forsythin in the Forsythia leaves:The single factor results showed that the concentration of Forsythin was the highest when the amount of water was 15 times.Forsythin was extracted at different temperatures,the highest concentration of Forsythin was obtained at 80℃.The concentration of Forsythin was the highest after four times of extraction.When extracting at different time,the concentration of Forsythin obtained with the extraction time of 40 min is the highest.Orthogonal test results showed that the best extraction conditions were 20 times the amount of water,two extractions at 90℃,60 min each time.(2)Preventive effect of Forsythin extract in Forsythia leaves on obesity induced by high-fat diet in rats:Compared with the blank control group,the weight of rats,Lee’s index,visceral fat content and its coefficients,TC,LDL-C,TBA,LP and FFA levels of the model control group were significantly increased(P<0.05).Hepatocytes were swollen,vacuolated and concentrated,hepatocytes were necrotic,liver lobules were unclear,hepatic cords were arranged disorderly,red blood cells appeared between hepatic cords,liver fatty liver pathology was obvious.The number of fat cells was reduced,fat was filled and cells were enlarged.And the equivalent diameter,circumference and area of adipocytes were significantly larger than that of the blank control group(P<0.05).After 6 weeks of Forsythin extract intervention,the medium and high dose group and standard group reduced the weight gain of rats induced by high-fat diet to a certain extent.It also reduced Lee’s index,visceral fat content,serum TC、LDL-C and LP levels.Among them,the high-dose group and the model control group were significantly different(P<0.05).There was no difference between the middle-dose group and the standard product group(P>0.05),and only the high-dose group can significantly reduce the serum FFA level(P<0.05).Under a 400-fold light microscope,the liver and fat morphology of each intervention group was improved.Compared with the model control group,there were less sinusoidal dilatation,loose cytoplasm,swelling of hepatocytes and round fat vacuoles in the intervention group.Among them,the low-dose group had the most severe symptoms of fatty liver,followed by the medium-dose group,and the high-dose group had the least severe symptoms.The fatty liver symptoms of the standard group were similar to those of the mediumdose group;the number of fat cells in each intervention group increased compared with the model control group.Adipocytes became smaller,but the effect of the low-dose group was not obvious,and the effect of the high-dose group was the most significant.Compared with the model control group,the area of adipocytes in the low-dose group was reduced.The difference was statistically significant(P<0.05).The circumference and area of adipocytes in medium dose group decreased,and the difference was statistically significant(P<0.05).The equivalent diameter,perimeter and area of adipocytes in the high-dose group were significantly reduced(P<0.05).The intervention effects of the standard product group and the medium-dose group were similar,showing that the number and size of adipocytes were similar.(3)Effects of Forsythin extract in Forsythia leaves on AMPK/ACC/SREBP-1c signaling pathway:The protein expression level of p-AMPK and p-ACC and pAMPK/AMPK and p-ACC/ACC in the model control group were significantly downregulated than those of the blank control group(P<0.05),and the protein expression level of SREBP-lc was significant up-regulated(P<0.05).Compared with the model control group,the expression levels of p-AMPK and p-ACC protein and pAMPK/AMPK and p-ACC/ACC in adipose tissue of obese rats in the medium-dose group,high-dose group and standard product group were significantly up-regulated(P<0.05).The high-dose group had the most obvious effect,which was similar to the normal.The middle-dose group and the standard product group are more similar.The expression levels of SREBP-lc and FAS protein in the medium and high dose group and the standard product group had a certain downward trend than those of the blank control group,and the trend in the high dose group was the most obvious,but the difference was not statistically significant(P>0.05).(4)Effects of Forsythin extract in Forsythia leaves on intestinal microflora and SCFAs content:The abundance of intestinal microbiota in the model control group decreased than those of the blank control group(P<0.05),and the diversity did not change significantly.At the phylum level,the abundances of Firmicutes,Bacteroidetes and Proteobacteria had no significant change,while the abundance of Verrucomicrobia was significantly increased(P<0.05).After the intervention of the high-dose group,the composition of the intestinal flora changed and was similar to the normal group.The ratio of G-bacteria to G+bacteria in the high-dose group and the middle-dose group decreased significantly than those of the model control group,but there was no statistical difference(P>0.05).At the genus level,the average relative abundance of Lactobacillus in the model control group was higher than the control group,but it was not statistically significant(P>0.05).Each dose group can reduce the abundance of Lactobacillus.The abundance of Ruminococcaceae_UCG-014 and Romboutsia in the model group decreased,while the relative abundance of Ruminococcaceae_UCG-014 and Romboutsia increased in the medium and high dose groups.The standard group could increase the abundance of Ruminococcaceae_UCG-014.However,for Ruminococcaceae_UCG-014,only the high-dose group showed statistical difference(P<0.05).For Romboutsia,only the medium-dose group showed significant difference(P<0.05).The abundance of Akkermansia in the model group was significantly increased than those of the blank control group(P<0.05),and each intervention group was able to reduce its abundance.However,only the difference between the high-dose group and the model group was statistically significant(P<0.05).The content of total short-chain fatty acid in model group was significantly higher than that in the normal group(P<0.05),and the content of total short-chain fatty acid in intervention groups was significantly lower than that in the model group(P<0.05).Conclusion:(1)The optimal extraction process conditions for Forsythin extract in Forsythia leaves were 20 times the amount of water,and extracting twice at 90℃ for 60 min each time.(2)Forsythin extract in Forsythia leaves can prevent obesity in rats induced by high-fat diet by controlling the increase of TC,LDL-C,LP and FFA levels in obese rats.Moreover,Forsythin extract can slow down the growth of adipose tissue cells,reduce the visceral fat content and its coefficient,Lee’s index and body weight of obese rats,and it can prevent the obesity induced by high-fat diet in rats.Forsythin in Forsythia leaves may play the major role in the effect.(3)Forsythin extract in Forsythia leaves can be used as a natural activator of AMPK to prevent obesity in rats induced by high-fat diet.Its efficacy may be mediated through activation of AMPK/ACC/SREBP-lc pathway.(4)Forsythin extract in Forsythia leaves can increase the abundance of intestinal microbiota,improve the structure of the intestinal flora of rats induced by high-fat diet,and make it normal.At the same time,it can also reduce the increasing trend of total SCFAs content in the intestinal tract of rats induced by high-fat diet,so as to promote the metabolism of SCFAs by intestinal microorganisms.