The Study on Antioxidation and Mechanism of 2-Bromo-4'- Nitroacetophenone

The 2-Bromo-4'-nitroacetophenone is an important compound which is widely used in the modification of methionine residues in alpha-chymotrypsin, organic synthesis materials and pharmaceutical intermediates. Our laboratory recently found that the 2-bromo-4'-nitroacetophenone can extend the lifespan of the C. elegans. Therefore, we want to know whether this compound has the anti-oxidative ability. In this paper, we used three different concentrations of 2-bromo-4'-nitroacetophenone to feed normal C. elegans and the mutants (daf-16). we found that 2-bromo-4'-nitroacetophenone has antioxidant effect and this effect is dependent on Insulin pathway.


Introduction
Upon stepping into the twenty-first century, how to solve the problem of aging is attracting more and more people's attention. The current majority of the country's population reached or exceeded the total population for more than 10 percent. With the lifting living standard, how to solve the problem of aging will be the arduous task facing mankind (Zhuang et al., 2016;Weiming Zhang et al., 2013) . From the biological point of view, aging is the decline of body structure and ability with the time passing by. From the pathological point of view, aging is due to the body's cell state emergency caused damage, resulting in the body's immune response to decline and metabolism ( Jiong Wu et al., 2017;Guo SH et al., 2007;).
The essence of aging is the process of making the various tissues and organs of the organism unable to meet the needs of the organism as the age increases. Although aging is the inevitable result of nature ( Nie GJet al., 2002, Gami MS &Wolkow CA, 2006, it is found that the process of aging can be effectively carried out by some biological means (Wilkinson DS et al., 2012;Sangha JS et al., 2012) . In this experiment, we used the C. elegans as a model organism to feed normal C. elegans and mutants with 2-bromo-4'-nitroacetophenone and found that 2-bromo-4'-Nitroacetophenone has antioxidant properties. The mechanism is mainly through the control of the insulin pathway to achieve.

Reagents and C. elegans Strain Preparation
The 2-bromo-4'-nitroacetophenone is obtained from Sigma-Aldrich (St. Louis, MO, USA), and its purities is 95%. It was dissolved in M9 solution and then diluted by M9.
Nematodes used were wild-type C. elegans N2, mutants of daf-16 originally obtained from Caenorhabditis Genetics Center. They were maintained on nematode growth medium (NGM) plates seeded with Escherichia coli OP50 at 20℃ as described.
Gravid nematodes were washed off the plates into centrifuge tubes, and were lysed with a bleaching mixture (0.45 M NaOH, 2% HOCl). Age synchronous populations of L1-larvae nematodes were obtained by the collection. Nematodes were washed with a modified S medium (Ren HR et al,2011;Brenner S, 1974) . Exposures were performed from L1-larvae to adult (prolonged exposure) in K medium of 96-well sterile tissue culture plates at 20℃ incubator in the presence of food. Paraquat was added forty eighteen hours later ( Donkin S & Williams PL, 2012).

Locomotion behavior
To determine the Locomotion behavior of lifespan. Head thrash and body bend were used as endpoints ( Akesson Chet al., 2003). The number of head wobbles and body bending times of Caenorhabditis elegans in the specified time were observed.
During the locomotion behavior assay, the examined nematodes were transferred into the assay plate containing K medium on top of the agar. After a recovery time of 1 min, head thrashes, and body bends were counted for 1 min and 20s respectively. (Kim J et al., 2008;Louppis A P et al., 2010).

Stress assy
To assay thermal stress resistance, worms pre-treated with the 2-bromo-4'-nitroacetophenone for 48-hr were transferred to the 35℃ condition, and then the lifespan was measured and analyzed as described above. To determine antioxidant activity, paraquat was added to Caenorhabditis elegans pre-treated with 2-bromo-4'-nitroacetophenone for 48 hours, and then the longevity was measured and analyzed as described above ( Honda Y et al., 1999;Sutphin GL et al., 2012) .

Statistical analysis
All data in this article are expressed as mean ± standard error (S.E.M.). Generate charts using Microsoft Excel (Microsoft Corporation, Redmond, WA). Statistical analysis was performed Journal of Biology and Life Science ISSN 2157-6076 2018 using SPSS 12.0 (SPSS Inc., Chicago, USA). Differences between groups were determined by analysis of variance (ANOVA). Probability levels 0.05 and 0.01 are considered statistically significant.

The 2-Bromo-4'-nitroacetophenone has improves the locomotion behavior of Caenorhabditis elegans.
We studied the effects of 2-bromo-4'-nitroacetophenone on the locomotion behavior of C. elegans and found that the nematode treated with 2-bromo-4'-nitroacetophenone significantly improved the motor behavior of C. elegans( Figure 2).

2-bromo-4'-nitroacetophenone has antioxidant effect and this effect is dependent on Insulin pathway
Compared with the control group, the mutants of C. elegans had significantly lower antioxidant activity, indicating that 2-bromo-4'-nitroacetophenone on Caenorhabditis elegans oxidation resistance through control of genes daf-16 to achieve. (Figure 4) (Xu X et al., 2007;.  ISSN 2157-6076 2018

5).
In summary, we can demonstrate that daf-16 participates in the antioxidant regulation of C. elegans by mutant daf-16, that is, the antioxidant activity of C. elegans is mainly controlled by genes. (Figure 4 and Figure 5 ).

Discussion
In this experiment, we found that 2-bromo-4'-nitroacetophenone had a significant antioxidant effect, and reached its best at 1ug/ml concentration [ Figure 1]. In addition, compared with the control group, the nematode treated with 2-bromo-4'-nitroacetophenone significantly improved the motility of the Caenorhabditis elegans during the aging process [ Figure 2]. These results indicate that 2-bromo-4 '-nitroacetophenone has a potential anti-oxidative effect.
Under the thermal stress conditions, the C.elegans treated with 2-bromo-4'-nitroacetophenone were able to survive for longer periods under the same conditions compared to the control group[ Figure 3), the condition of 2-bromo-4 '-nitroacetophenone has the effect of improving heat stess.
As can be seen from Figure 4, under the same conditions, the survival rate of daf-16 nematode is reduced. From the above data, the compound 2-bromo-4'-nitroacetophenone Control of nematode life is achieved mainly through the control of the corresponding genes, in which daf -16 nematode life is of the greatest impact. That is to say 2-bromo-4'-nitroacetophenone has antioxidant effect and this effect is dependent on Insulin pathway.
Correspondingly, after the mutation of the C. elegans, its movement behavior will be correspondingly reduced, and these data further confirmed our conclusion. However, the mechanism of controlling the life of nematodes is very complex and requires more experiments to verify.