| Hawthorn(Crataegus spp)belongs to the germs Crataegus of Rosaceae which is native to China.It is one of traditional common medicinal and food materials with history of more than 2000 years.Hebei Province is the main production area of hawthorn in China.The fruit of hawthorn is rich in a variety of bioactive substances,such as polyphenols,polysaccharides,flavonoids,organic acids and so on.Among them,procyanidins is one of the active substances with high content in hawthorn.Due to the complex composition and unclear digestive characteristics,it is difficult to clarify its mechanism of procyanidins.In addition,the antioxidant activity of procyanidins has been recognized,but there are few reports on the regulation of lipid metabolism disorders.The obesity,cardiovascular and cerebrovascular diseases,diabetes and other chronic diseases caused by lipid metabolism disorders have become global health killer.Therefore,our study explored the dynamic changes of HPC in the process of digestion and fermentation,as well as the interaction with gut microbiota through in vitro simulated digestion and fermentation experiments,then systematically studied the improvement effect of HPC on rats with lipid metabolism disorder,and tried to clarify the possible biochemical and molecular mechanism.At the same time,we further explored and proved the improvement effect and mechanism of HPC on liver lipid accumulation through HepG2 cells,and conducted a more in-depth study on its mechanism.The main results obtained in this study are as follows:(1)Study on the dynamic changes of hawthorn procyanidins during in vitro digestion and gut metabolism.Procyanidins are a kind of macromolecular compounds with low digestibility and absorption rate.Their final potential biological activity in vivo mainly depends on their metabolism,absorption and distribution after intake.In order to investigate the relationship between the bioactivity and bioavailability of HPC,this study first analyzed the basic structure of HPC.On this basis,the dynamic changes of HPC in the digestion track and the interaction between gut microbiota and HPC were studied by in vitro digestion and fermentation model.The results showed that the content of procyanidin in hawthorn procyanidin extract was 81.85±1.05 mg/100 mg,which was mainly composed of four flavane-3-alcohols:epicatechin EC(36.12%),procyanidin B2(10.06%),procyanidin B5(7.49%)and procyanidin C1(5.03%).The results of in vitro simulated digestion showed that the stability of hawthorn procyanidins in oral digestion was high;After 6 hours of gastric digestion,the content of total procyanidins(1.473 mg/mL)decreased significantly(P<0.05);And continuously decreased to 1.08 mg/mL in intestinal digestion(P<0.05).In the whole process of in vitro digestion,the total content of procyanidins decreased by 12.02%.The contents of EC,procyanidin B2,procyanidin B5 and procyanidin C1 remained stable after oral digestion;After gastric digestion,procyanidin B2,procyanidin B5 and procyanidin Cl decreased significantly by 22.97%,69.65%and 20.87%respectively compared with oral digestion stage(P<0.05),while EC increased significantly by 10.83%(P<0.05);After intestinal digestion,EC increased from 39.93%in gastric phase to 41.73%(P<0.05),while the contents of procyanidin B2,procyanidin B5 and procyanidin Cl decreased by 14.27%,49.17%and 10.48%,respectively.As HPC mainly reach the colon and are degraded and utilized by intestinal microbiota,the pH value of human intestinal microbial fermentation broth decreases with the production of degradation products,and the total procyanidins also decreases.16sDNA sequencing showed that HPC could regulate the intestinal microbial population,mainly down regulate the abundance of Bacteroides,Fusobacterium,Enterococcus,Parabacteroides and Bilophila,and up regulate the abundance of Escherichia Shigella,Klebsiella,Turicibacter,Actinobacillus,Roseburia and Blautia;Metabolomic analysis showed that EC,procyanidin B2,procyanidin B5 and procyanidin C1 could be transformed into phenolic acids through the metabolism of Bacteroides,Sutterella,Butyrobacter and Blautia.4-ethylbenzoic acid,4-hydroxyphenylpropionic acid and 3,4-dihydroxyphenylacetic acid were also identified as important metabolites in the fermentation of hawthorn procyanidins;At the same time,through the enrichment of differential metabolites,we found that some differential metabolites were annotated into AMPK signaling pathway.These results explained the potential mechanism of HPC metabolism,and the changes of gut microbiota and colonic phenolic acid suggested that HPC played a positive regulatory role in the gut by regulating AMPK signaling pathway.(2)Effect and mechanism of HPC on alleviating lipid metabolism disorder induced by high-fat diet.After clarifying that HPC have a positive effect on improving gut microbiota and might regulate AMPK signal pathway,we established the lipid metabolism disorder induced by high-fat diet rat model to study the improvement effect and mechanism of HPC on lipid metabolism disorder.The results showed that HPC could significantly reduce the lipid levels of serum and liver in lipid metabolism disorder rats(P<0.05)and improve the structure of liver tissue.At the same time,HPC could inhibit the occurrence of insulin resistance and oxidative stress caused by high-fat diet.In addition,we found that HPC could regulate the structure of gut microbiota,especially reverse the decrease of Akkermansia,Bacteroides and Adlercreutzia,as well as the increase of Lactobacillus,Bifidobacterium,Blautia,Lachnospiraceae and Subdoligranulum,and increase the production of SCFAs.Importantly,the results showed that the disturbance of gut microbiota caused by high-fat diet led to the increase of LPS level,and the intervention of HPC could reduce the leakage of LPS by enhancing the function of intestinal barrier(occluding and claudin-1).HPC further inhibited TLR4 and NF-κB,MyD88,IKKβ,and downstream IL-1 β,TNF-α in small intestine and liver.The expressions of key proteins could reduce hepatic inflammation and restore liver lipid metabolism.At the same time,the results of metabolomics showed that the related differential metabolites after HPC intervention were enriched and annotated into AMPK signaling pathway.The results of animal experiments showed that HPC intervention could regulate the related proteins of AMPK signaling pathway,reducing the lipid accumulation in hepatic cells and alleviating the disorder of lipid metabolism,which showed that HPC might play a crucial role in intervening lipid metabolism disorder by mediating gut liver axis,which further confirmed the prediction results of metabolomics on signal pathway.(3)The mechanism of HPC on alleviating lipid accumulation induced by oleic acid in HepG2 cells.It is verified that HPC could improve the lipid accumulation of HepG2 cells by regulating AMPK signal pathway in the lipid metabolism disorder model of HepG2 cells induced by oleic acid,and further revealed the molecular mechanism of HPC intervening in lipid metabolism disorder.The results showed that HPC treatment could reduce the contents of TG and lipid droplets in HepG2 cells induced by oleic acid(P<0.05),so as to improve the lipid accumulation of HepG2 cells induced by oleic acid in a dose-dependent manner.At the same time,high-dose HPC could also significantly reduce the increase of ROS level induced by oleic acid(P<0.05).Western blot showed that HPC could improve lipid metabolism in HepG2 cells by up regulating p-AMPK and p-ACC(P<0.05)and down regulating the expression of SREBP-lc(P<0.05).In order to further verify the above results,the cells were treated with AMPK inhibitor dorsomorphin dihydrochloride.The results showed that the protective effect of HPC on HepG2 cells induced by oleic acid was weakened,meanwhile,the intracellular TG level continued to increase,the phosphorylation levels of AMPK and ACC proteins decreased significantly,and the expression of SREBP-lc protein increased significantly,It showed that AMPK inhibitor eliminated the protective effect of oleic acid induced HepG2 cells and the activation of AMPK pathway.Therefore,the intervention of HPC on lipid metabolism disorder is closely related to the activation of AMPK pathway. |
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