The Research On Establishing In Vivo Screening Platform Of Anti-fat Accumulation Natural Product Based On The Circuit Of "Peripheral CTSL-Central 5-HT"

Excess dietary energy intake leads to the accumulation of fat in the body,which is a primary cause of obesity and related metabolic diseases.Therefore,the study on the regulation mechanisms of fat metabolism using model animal and establishing a screening platform for natural food products to defense against obesity based on these mechanisms can not only provide the theoretical basis but also serve as new ideas and screening methods for food research against obesity and related metabolic diseases.Lysosomes and inside hydrolases have been regarded as "scavengers" in cells,involved in the degradation and recycling of intra-and extracellular macromolecules and non-functional organelles.Recently,it has been reported that the lysosome,as a nutrient-sensing and lipophagy center,critically controls cellular homeostasis.However,the current research about lysosome on nutrient sensing is mainly under the condition of starvation in cells,working as a center of nutrition sensing and fat phagocytosis to regulate the lipid catabolism of cells.The regulation of lysosome and inside hydrolases on lipid deposition under the condition of excess nutrition in the whole body is unclear.Therefore,in this thesis,we explore the regulation mechanism of cathepsin L(CTSL)on fat metabolism in two model animals,Caenorhabditis elegans(C.elegans)and mice,and establish a vivo screening platform for natural products against fat deposition based on the related mechanism.This thesis can be divided into three parts.In the first part,we explore a conserved"Peripheral CTSL-Central serotonin(5-HT)" circuit regulating fat accumulation in both C.elegans and mice starting from lysosome nutrition sense.In this part,we firstly established a model of fat accumulation in C.elegans using glucose and palmitic acid supplementation in a short time.In this fat accumulation model,we confirmed that the supplementation of nutrients did not affect the development of C.elegans.Further,we found that lysosomal biosynthesis increased along with the supplementation of nutrition.And lysosomal inhibitor treatment could significantly reduce the basal and nutrient-induced fat accumulation of C.elegans.These results suggested that lysosomal nutrient sensing regulated the fat accumulation of C.elegans.Next,we carefully studied the expression of lysosomal cathepsins induced by nutrients supplementation and found that the expression of mammal CTSL homologous gene cpl-1 increased in a dose-dependent manner.While the inactivation of cpl-1 by RNAi and mutation can significantly reduce the fat accumulation and lysosome biosynthesis,these results suggested that CPL-1 was required for the regulation of lysosome biosynthesis and fat accumulation in C.elegans.Then,we found that the inactivation of cpl-1 enhanced the level of 5-HT,the genes expression of 5-HT signaling,and 5-HT related behaviors,using fat regulation related mutants of C.elegans.Tissue-specific cpl-1 RNAi of peripheral fat storage tissues such as intestine and hypodermis significantly increased the expression of 5-HT synthesis gene tph-1 in C.elegans ADF neurons.Meanwhile,mod-1 and ser-6 mutants,5-HT signal receptors,significantly reversed the decrease of fat accumulation caused by cpl-1 RNAi,which indicated a fat regulation circuit of"Peripheral CPL-1-Central 5-HT" in C.elegans.Finally,using ctsl gene knockout mice and CTSL inhibitor,we found that the circuit of "Peripheral CTSL-Central 5-HT" also conservatively regulated WAT fat deposition,finally,affected the diet-induced obesity of mice.In the second part of the thesis,we studied the effects of dietary flavonoids,such as luteolin(LU),on fat regulation of C.elegans at basic level,and found that LU could reduce the fat accumulation of C.elegans through the circuit of "peripheral CPL-1-Central 5-HT".According to our previous reports,dietary LU reduces the expression and activity of CTSL in adipose tissue and improves diet-induced obesity in mice.To clarify the relationship between the effects of LU and the circuit of "Peripheral CTSL-Central 5-HT",we evaluated 6 kinds of compounds,including LU,chrysin,apigenin,quercetin,kaempferol and myricetin,which are common in the diet based on the backbone of 5,7-dihydroxylflavone.We found all of these flavonoids had inhibitory effects on C.elegans fat accumulation at 100 ?M.While,only LU had inhibitory activity on C.elegans fat accumulation at 25 ?M,suggesting that LU may inhibit C.elegans fat accumulation through some distinct mechanism.Further,we found that tph-1 mutation could ultimately reverse the inhibition of C.elegans fat accumulation induced by LU.The treatment of LU enhanced the expression of tph-1,mod-1,and ser-6.Meanwhile,the mutation of mod-1 and ser-6 could also reverse the inhibition of C elegans fat accumulation induced by LU.However,those as mentioned above,5-HT signal-related changes did not occur in C.elegans treated with other 5 flavonoids,indicating that LU can inhibit fat accumulation by activating C.elegans 5-HT signal.Finally,we also confirmed that LU treatment could increase the expression of tph-1 in ADF neurons and promote lipolysis and fatty acid ?-oxidation of C.elegans.In the third part,based on the study of the above two sections,we analyzed the effects of more than 24 kinds of natural products on fat regulation induced by short-term glucose supplementation in C.elegans.We established the in vivo screening platform for natural products regulating fat deposition.In this part,we crossed CPL-1::mChOint worm with TPH-1::GFP worm and established a double fluorescence expression system to screen the possible natural products regulating fat deposition.Further,based on the circuit of "Peripheral CPL-1-Central 5-HT",we analyzed the effects of more than 24 kinds of natural products on C.elegans fat accumulation using the dual-fluorescence system.We monitored the expression of CPL-1 and TPH-1 along with the fat deposition induced by the treatment of 24 kinds of natural products.We found that 10 kinds of natural products such as daidzin could reduce the fat accumulation in the condition of short-term glucose supplementation in C.elegans through the circuit of "Peripheral CPL-l-Central 5-HT".Therefore,the C.elegans dual-fluorescence system based on the "Peripheral CPL-1-Central 5-HT" circuit could be used as a rapid and efficient in vivo screening platform for screening fat regulation natural products.In summary,this thesis we uncovered a novel conservative fat regulation circuit"Peripheral CTSL-Central 5-HT" using the model organisms of C.elegans and mice.Based on this conserved circuit,we revealed the mechanism of LU and other dietary flavonoids in the fat regulation of C.elegans.We esbalished an in vivo screening platform for natural products based on this above circuit.This study will provide new ideas and technical methods for food nutrition research on obesity and related metabolic diseases.