| Sargassum fusiforme is reputed as "longevity promoting vegetable",and the Sargassum fusiforme polysaccharides were reported with anti-oxidant and anti-aging activities.However,the underlying mechanisms how Sargassum fusiforme polysaccharides exert antioxidant and anti-aging effects remain unclear.Jnk(c-Jun N-terminal kinase)was proposed as a longevity gene,and our previous study has found a significant increasing expression level of Jnk in the livers of male mice with long-term SFPS treatment.So,how does the SFPS perform antioxidant and anti-aging effects by modulating the expression patterns of Jnks?Overexpress JNK in fruit flies and nematodes can significantly prolonged the lifespan by enhancing the body's antioxidant capacity.However,in mammals,the JNKs derive three major homolog genes,Jnk1,Jnk2 and Jnk3(Jnk3 is mainly expressed in the heart,testes and brain,whereas Jnk1 and Jnk2 are widely expressed in the body).The connection between the levels of Jnk expression to aging is still not clear.Real-time quantitative PCR(qPCR)and Western Blot were used to detect the expression patterns of JNK1/2 in the liver of different months(3 months,9 months,11 months and 20 months)male mice.The results showed that the mRNA and protein expression of JNK1 and JNK2 were significantly decreased with the increase of mice's age,that is,JNK1/2 was negatively correlated with aging in male mice.At the same time,JNK1/2 with 54 kDa molecular weight had the largest change with the progress of aging.Subsequently,RT-PCR was used to specifically differentiate each isoform of JNK and examined the expression pattern of each isoform in normal mouse.The results showed that the mRNA expression of Jnk1-?2,Jnk2-?1 and Jnk2-?2 were significantly down-regulated as the age of mice was increased(JNK2-?1 is translated into 46 kDa protein and JNK2-?2 are translated into 54 kDa protein).Therefore,our study suggests that Jnk1-?2,Jnk2-?1 and Jnk2-?2 play an important role in the process of aging.So how does SFPS affect the expression pattern of JNK? Our study showed that the protein expression levels of JNK1/2 were significantly up-regulated in the livers of 20-month-old mice which fed with SFPS.Futhermore,the up-regulation degree of JNK 54 kDa protein was much higher than the JNK 46 kDa protein.Moreover,the mRNA expression level of Jnk1-?2(JNK1-?2 translated as 54 kDa protein)showed the most significant change.Therefore,we speculated that SFPS was closely related to the Jnk1-?2 expression level.Then JNK's protein structure were predicted and JNK's bioinformatics were analysised,the results showed that JNK protein tertiary structure is similar to each isoforms and the different sequence lies in the subdomain.The C-tail alternative splicing at the C-terminal tail results in long(54kDa)and short(46kDa)JNK variants.Active loop is located in the subdomain IX and X which reflect the difference of each isoform,and it is an important site for JNK to identify the substrate.Both of Active loop sequence and the C-terminal tail play an important role in the specific reaction between JNK and the substrates.Multiple sites on antioxidant regulatory factor NRF2 are same as the conserved sites of JNK's substrates,suggesting that NRF2 may be a substrate of JNK.Co-IP experiments results showed that the 54 kDa JNK protein not the 46 kDa JNK interacts with NRF2,then activates the NRF2 / ARE antioxidant signaling pathway.In summary,long-term feeding SFPS can effectively promote the expression of Jnk1-?2(54kDa)in old mice,and enhance the interaction between JNK1-?2 and NRF2,promote NRF2 into the nucleus then enhance the expression of NRF2 downstream enzyme gene which are related to antioxidant and detoxification.Thereby the body's overall antioxidant capacity was enhanced,and ultimately delay the aging process in mice. |
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