| Aging is characterized by widespread but complicated degenerative change in most if not all tissues, which is influenced by both genetic and environmental factors. Great progresses have been made in the research on aging related genes in recent years, but the progresses on identifying molecules of therapeutical potential for delaying aging are quite limited.According to the oxidative damage theory, free radicals, particularly reactive oxygen species (ROS) such as superoxide, hydrogen peroxide and hydroxyl radical, are one of the major factors in the aging process, and they are associated with many diseases such as heart disease and cataract. This theory predicts that augmentation of defenses against ROS by antioxidants should retard aging. This prediction has been tested many times over the years, in Caenorhabditis elegans, in Drosophila melanogaster, and in mammal animals.Peroxidases are an important class of iron porphyrin-containing enzymes, they catalyze substrate oxidations using hydrogen peroxide, but the therapeutical potential of natural peroxidases is severely limited by their instability, scarce solubility, cost, and other factors. The heme-peptide complexes from hydrolytic digestion of cytochrome C exhibit peroxidase activity, and they are called microperoxidases such as microperoxidase-8 (MP-8), microperoxidase-11 (MP-11), and microperoxidase-9 (MP-9). These microperoxidases are smaller than natural peroxidases, but they also have a number of significant disadvantages. First, their specific covalent binding makes chemical synthesis difficult, very likely due to the poor affinity between peptides and hemin. Second, the resulted and hemin-coupled peptide mimetics are generally unstable. Third, they usually have poor water solubility. In our previous work, we designed a novel peroxidase mimics Deuterohaemin- Ala-His-Thr-Val-Glu-Lys (DhHP-6) and investigated its anti-cataract activity and peroxidase activity in vitro. The in vitro peroxidase activity of DhHP-6 is very similar to that of MP-11, but DhHP-6 has very good water solubility, stability and its chemical synthesis is much simpler.In the present investigation, we used the model organism Caenorhabditis elegans (C.elegans) to evaluate the pharmacological effects of DhHP-6 on aging and examine their molecular mechanisms. C. elegans is a popular model for studying aging and longevity due to its short lifespan and the well defined genetic and environmental factors affecting lifespan.Treatment of wild-type C. elegans by DhHP-6 significantly increased its lifespan by 20% and delayed the age-related decline of fast body movement in our laboratory culture condition at 20℃, but it had no effect on the decline of pharyngeal pumping and self-fertile reproduction. These results shown that DhHP-6 extend the life stageⅡ, the period characterized by vigorous motor activity. DhHP-6 did not extend the life span of wild type worms further in the media that bacterial growth was arrested by ampicillin and in the axenic liquid media, and it did not arrest the growth of E.coli OP50, the food of the C.elegans either, showed that it may act in the calorie restriction (CR) signal pathway but not by restricting the worms'food source.Based on the technique of double-stranded RNA interference (RNAi), we successfully established the model of rapid aging in C.elegans by reducing the expression of ctl-2 gene. The inhibition of the gene of ctl-2 by RNAi shows the phenotypes of short life span and the decline of self-fertile reproduction. We find that DhHP-6 can extend the maximum of life span of normal nematode and it can delay the rapid aging process. These findings suggest that DhHP-6 has the anti- oxidative damage activity in vivo and this activity can promote the life span of C.elegans.Further more, DhHP-6 can protect worms from acute heat stress and acute oxidative stress after two days administration. However, considering with the delaying protection of stress injury from DhHP-6, one reasonable hypothesis is that the increasing of life span and stress resistance mainly comes from the elevation of total endogenous antioxidant enzyme capacity by treatment of DhHP-6 but not the direct exogenous antioxidative effect of DhHP-6. In the previous research we found that DhHP-6 show good activities of scavenging hydroxyl free radicals (93% of microperoxidase-11) in vitro, but in the homogenate fraction the activity of SOD and catalase are stronger than that in vitro and also stronger than that in the homogenate fraction of the control group (without treatment of drugs).To further determine how DhHP-6 prolonged C. elegans lifespan, we analyzed the genetic requirements for these effects. The study of short-lived mutant animals has provided valuable insights into the mechanisms that shorten lifespan of organisms. We tested the effect of DhHP-6 upon longevity and aging using the daf-16 (mgDf50), sir-2.1 (ok434) and mev-1 (kn1) null mutation strains of C.elegans. We showed that treatment with DhHP-6 contributes to the shortened lifespan of worms lacking mev-1 gene, although it still can not restore the lifespan to that of wild type, consistent with the finding that DhHP-6 restored the life span due to the amelioration of oxidative stress. On the other hand, DhHP-6 treatment could not prolong lifespan of animals with defects in the daf-16 and sir-2.1 gene. One possible explanation of this founding is that the effects of DhHP-6 treatment are mediated, at least in part, through these genes. According to the report of Berdichevsky, both daf-16 and sir-2.1 gene regulate in the same signal pathway in C.elegans. On the one hand, sir-2.1 gene is regulated by daf-16 gene. One the other hand, SIR-2.1 can bind to DAF-16 by some unknown reason and active the activity of DAF-16 to affect the down stream gene and affect the life span and stress resistance. We propose that DhHP-6 beneficial effects through interaction with the same signal pathway regulated by these two genes.A number of genes have statistically significant changes in the transcript levels during aging in the C.elegans. Using real-time PCR, We examined the effect of DhHP-6 on these aging-related genes. DhHP-6 treatment blocked the increase of the expression of the hsps, and the decrease of sir-2.1 and sir-2.3. It could also increase the express of ctl-1, ctl-2 and sod-3 but no effect on the expression of ins-17, ins-18 and TC3 transposase family genes. These results indicate that these compounds had direct or indirect effects on these gene expressions.To further determine the relationship between the catalytic activity in vitro and the effect of DhHP-6 on extending life span, we had synthesized a novel compound with deuterohaemin but no histone (Deuterohaemin-Ala-Ala-Thr- Val-Glu-Lys, DhAP-6). Treatment with DhAP-6 can not extend the life span of wild type C.elegans and can not increase the catalase activity in vivo either. These results show that the effect of DhHP-6 on retarding aging based on the effect of scavenging free radicals in vitro.In conclusion, this study successfully established the model of rapid aging in C.elegans by reducing the expression of ctl-2 gene, based on the technique of double-stranded RNA interference (RNAi), systemically clarifies the effects of DhHP-6 on retarding aging and its physiological character. The study also intends to look insight the mechanisms of DhHP-6 in the regulation of aging signal pathway. Data presented in this thesis have provided a basis and a framework for further investigation towards the precise roles of the molecular mechanism of aging and this may shed light on the therapies of aging-related diseases.
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