Using Nematode Model To Study The Action Mechanism Of Low Molecular Weight Protein Tyrosine Phosphatase On Stress Response And Screening Of Anti-stress Natural Products

Low molecular weight protein tyrosine phosphatase(LMW-PTP)is a structural dysfunctionally protein tyrosine phosphatases(PTPs),which is separately divided into one type of PTPs family.LMW-PTP is highly conserved in evolution and exists in almost all organisms,including plants,prokaryotes,and archaea.Natural products are abundant in nature.They are important drug storehouses and have been extensively studied in the field of drug development.Nematode is an excellent model organism with strong reproductive ability,short life cycle,small body and easy to operate,transparent body and easy to observe,long-term cryopreservation in liquid nitrogen,and complete determination of cell lineage.At the same time,C.elegans are highly conserved in many important signaling pathways,with 60% to 80% homology with human genes.The C.elegans’ lmwptp gene is homologous to human acp1 gene.The computer structure simulation shows that the tertiary structure of the two proteins is very similar,so it is speculated that the biological functions and action mechanisms of the two have similarities.Stress response is a general term for the response of organisms to external stimuli.With the deterioration of natural environment and the acceleration of life rhythm,the types and frequencies of stimuli in vivo and in vitro are gradually increasing.These stimuli will destroy the balance between oxidation and antioxidant systems,affect the daily behavior and health of organisms,and lead to the occurrence of many diseases.Therefore,the prevention and treatment of biological anti-stress has been paid more and more attention.This paper used C.elegans model to study the action mechanism of LMW-PTP on stress response,and the anti-stress natural products were screened.Firstly,the pPD129.36-lmwptp plasmid for C.elegans’ lmwptp RNAi was successfully constructed and induced to express,and the LMW-PTP polyclonal antibody was successfully prepared by immunizing rabbits.The validity of lmwptp RNAi was confirmed by qPCR and Western Blot.Secondly,we examined the effect of lmwptp RNAi on the general physiological indexes of C.elegans.It was found that lmwptp RNAi did not affect the lifespan,spawning number,swallowing rate,body length of C.elegans,but the C.elegans on the 7th day after lmwptp RNAi showed a special state of motion.In addition to sinusoidal motion on the medium plane,C.elegans also exhibited “head-up” and/or “tail-up” motion in the vertical direction.We also found that the fat content of C.elegans increased significantly after lmwptp RNAi.This phenomenon was related to reducing the expression of some lipolytic genes,such as acdh-1,acs-2 and increasing the expression of some fat synthesis genes,such as fat-6,mdt-15,pod-2,elo-5 in the C.elegans after lmwptp RNAi.We further found that lmwptp RNAi significantly increased the stress resistance of C.elegans,including 35°C heat shock stress resistance,UV irradiation stress resistance,oxidation stress resistance.Then,we further explored action mechanism of LMW-PTP on 35°C heat stress.lmwptp RNAi was found to reduce ROS levels,increase SOD activity and reduce the accumulation of lipofuscin in C.elegans.LMW-PTP negatively regulated the stress resistance of C.elegans through insulin/IGF-1-like signaling pathway.LMW-PTP might act on DAF-2 to further change the nuclear translocalization of transcription factors DAF-16 and HSF-1 and the expression of their downstream target genes.Next,we selected 50 kinds of natural products to screen for anti-UV irradiation using C.elegans model.Because the effect of mint aqueous extract on UV irradiation resistance was stable,we further explored the reason why mint aqueous extract enhanced the UV irradiation resistance of C.elegans.We found that mint aqueous extract could reduce the ROS levels and increase SOD activity in C.elegans after UV irradiation,and this effect was related to the insulin/IGF-1-like signaling pathway.Mint aqueous extract could promote the nuclear translocalization of DAF-16 and promote the expression levels of its downstream target genes sod-3,hsp-12.6,hsp-16.1,hsp-16.49.Finally,we explored the role and mechanism of the aqueous component of mint,didymin,in the process of UV irradiation stress resistance.We found that 0.1 mM didymin could prolong the lifespan of C.elegans and improve the resistance of C.elegans to UV irradiation,35°C heat shock and juglone oxidative stress.Didymin could reduce ROS levels and increase SOD activity after UV irradiation.The effect of anti-UV irradiation of didymin in C.elegans was related to the insulin/IGF-1-like signaling pathway and might act on DAF-2 upstream of the pathway.Thereby,the nuclear translocalization of DAF-16 was promoted and the expression levels of its downstream genes sod-3 and hsp-16.2 were increased.In summary,we studied the biological function of LMW-PTP by using C.elegans model and explored its action mechanism in stress response.We also screened the anti-UV irradiation function of 50 kinds of natural products aqueous extract by using C.elegans model and focused on the action mechanism of mint aqueous extract and its aqueous component didymin to improve UV irradiation resistance of C.elegans.We not only discovered new functions of the PTPs family,but also provided a theoretical basis for the development of drugs for stress-related diseases.It had certain market prospects and social significance.