| With the acceleration of life and the intensification of environmental pollution,humans tend to be a sub-health state caused by the high pressure.In addition,the changes of diet structure,ultraviolet radiation,and chemical drug abuse have serious impact on the body.As a result,the body is insensitive to heal the oxidative damage and unable to resist the invasion of superoxide,then the inflammation,aging,and disordered metabolic regulation mechanisms will occur,finally leading to gene mutations and cancer.The research on the treatment of diseases caused by the peroxidation of the organism,has become the new demands of human health.Another important aspect was that the accumulation of heavy metals endangers human beings,animals and plants,which needs to be solved urgently in the environment.Metallothionein(MT)is a metal-binding protein with the high content of cysteine and has a relatively low molecular weight(6-7k Da).MT are existed widely in the biological world,and its special structure makes it own the ability to combine with metal ions,including cadmium,zinc,lead,iron and so on.Through the combination with metal ions,MT could reduce the toxicity of heavy metals and regulate the physiological state of the body.Previous studies showed that MT also has the function of scavenging reactive oxygen species(ROS),but the mechanism of that is still unclear.It is generally believed that it could scavenge the free radicals in the body to reducing oxidative damage.Researchers found the expression of MT in various malignant tumors and MT exerted dual functions of promoting or suppressing cancer through various mechanisms.Safety evaluation is the basis and guarantee of application.With the development of MT research,toxicity evaluation is far behind its application,development and function.In addition,the emergence of new preparation methods of MT makes the product itself more diversified.In particular,the sample rh-MT-Ⅲαwe selected in this research is a new substance produced by genetic engineering,which specific toxicity effect and mechanism are not clearly defined.Although the original dilemma could be solved by rh-MT-Ⅲα,there is a lack of toxicity and functional evaluation of rh-MT-Ⅲα.Further studies concerned with this promising area are urgently required.Before conducting this experimental research,we explored the relevant applications and effects of MT in the field of medicine and cosmetics through literature retrieval and analysis.According to the feedback data of MT in laboratories,we found that the mainly acquisition types of MT was the extraction from the animal or plant tissue with low purity and lack of studies on the toxicity evaluation.According to research progress of MT in laboratory and product conditions in market,this study selected recombinant human metallothionein-Ⅲαpeptide(rh-MT-Ⅲα)as the research object.Firstly,the safety of rh-MT-Ⅲαwas investigated by cells and C.elegans models.In the aspect of function evaluations,chemical co-incubation was used to explore the free radical scavenging rate of rh-MT-Ⅲαin vitro.Human immortalized epidermal cells(Ha Ca T)were selected to establish the UVB damage model and the availability of that model was investigated by MT which were purificated from rabbit liver.The Ha Ca T cells were treated with UVB and rh-MT-Ⅲαat different doses to evaluate the antioxidant function of rh-MT-Ⅲα.The C.elegans were treated with Ag NPs and rh-MT-Ⅲαat different doses to evaluate the relief function of rh-MT-Ⅲαon C.elegans induced by Ag NPs.The main conclusions were as follows:1.Cytotoxicity evaluation of rh-MT-Ⅲα:Human immortalized keratinocytes(Ha Ca T)and L929 mouse fibroblast cells(L929)were selected to be treated with rh-MT-Ⅲαat different doses(25～400μg/m L).After 24 hours of exposure,the indexes of cytotoxicity were detected,including viability,LDH release,morphology,apoptosis of cells.(1)The results of cells viability showed that,compared with the control group,both the two cells viability had obvious change in the experimental group.At 400μg/m L,the viability of the two cells decreased,but these changes were not significant.(2)The results of cell morphology showed that,compared with the control group at the highest dose(400μg/m L),rh-MT-Ⅲαhad no significant influence on the two cells morphology.(3)The results of LDH release showed that,compared with the control group,the LDH release of the two cells increased significantly at dose of 400μg/m L,but there was no significant difference at dose of25～200μg/m L.(4)The results of cell apoptosis rate showed that,compared with the control group,apoptosis rates of the two cells increased significantly at dose of 400μg/m L,especially the early apoptosis rate,which was 4 to 5 times that of the control group.Those results showed that rh-MT-Ⅲαhas no obvious toxicity to the two cells under the experimental dose of 400μg/m L.2.Evaluation of the general toxicity of rh-MT-Ⅲαto C.elegans:After exposed to the rh-MT-Ⅲαat doses of 5,50,500μg/m L for 48 hours,the general toxicity evaluation index of C.elegans were detected,including movement(head swing frequency and body bending frequency),lifespan,growth(length and width),feeding(pumping frequency)and reproductive function evaluation(number of offspring).The results showed that,compared with the control group,each biological endpoint showed no significant difference,indicating that rh-MT-Ⅲαhad no toxicity on C.elegans at the experimental dose.3.Antioxidant function study on rh-MT-Ⅲα:(1)Free radical scavenging in vitro:1.5 m L rh-MT-Ⅲαwas mixed with 1.5 m L 1,1-diphenyl-2-picranil(DPPH,80μg/m L)solution in vitro.After that the free radical scavenging rate were measured and the IC50(rh-MT-Ⅲαconcentration required when DPPH scavenging rate reaches50%)was calculated by the data.The results showed that the free radical scavenging rate of rh-MT-Ⅲαincreased with the increase of rh-MT-Ⅲαdose,and the IC50 was184.32μg/m L.(2)Model construction of Ha Ca T cells injury induced by UVB:Ha Ca T cells were treated with UVB at a certain radiation intensity(100μw/cm~2)and different radiation time(1 min,2 min,3 min,4 min,5 min),then we detected the cell viability(%)and the result were 99.27±0.11,94.34±0.03,59.39±12.03,39.80±8.75,8.95±0.68.According to the results,we selected the radiation time of 3 min to ensure that(1)UVB radiation could cause oxidative damage to Ha Ca T cells;(2)it could have biological significance in the recovery of the viability of the Ha Ca T cells after the UVB exposure.The model was evaluated by MT from rabbit liver and the results showed anti-oxidative effect at a dose of 75μg/m L,indicating that the model was effective.(3)Antioxidant function study:After the UVB exposure,the Ha Ca T cells were treated with rh-MT-Ⅲαat different doses(25,50,100,200μg/m L)for 24 hours.The results showed that,compared with the negative control group,the cells viability was decreased in the UVB treatment group,while the cells viability showed an increase trend at a dose of 25～100μg/m L in the rh-MT-Ⅲαtreatment groups,then decreased at 200μg/m L dose.Meanwhile,there was no significant difference at dose of 50,100μg/m L between negative control group and rh-MT-Ⅲαtreatment groups.The cells viability was significant higher than positive control group at 100μg/m L.The results of cells morphology,apoptosis,and reactive oxygen(ROS)content were consistent with the cell viability.Compared with the positive group,the cells were more tightly connected,and the apoptosis rate and ROS content were significantly reduced in the rh-MT-Ⅲαdose group at 100μg/m L.Those results showed that rh-MT-Ⅲαhad the function of anti-oxidation on oxidative damage of Ha Ca T cells induced by UVB and it was more effective at dose of 100μg/m L.4.Relief effect of rh-MT-Ⅲαon toxicity to C.elegans induced by nanosilver:The preliminary study showed that nanosilver(Ag NPs)has obvious toxicity to C.elegans and can cause oxidative damage to nematodes at a dose of 10μg/m L.In this chapter,Ag NPs were used as a positive control at a dose of 10μg/m L.The C.elegans were treated with 10μg/m L Ag NPs solution and different concentrations of rh-MT-Ⅲα(5,50,500μg/m L)at the same time for 48 hours,and the movement growth and ROS indexes were detected after the exposure to investigate the alleviation function of rh-MT-Ⅲαon Ag NPs toxicity to C.elegans.(1)The results of movement showed that,compared with the control group,the frequency of head swing(/20 s)and body bending(/60 s)of the C.elegans were significantly reduced in the Ag NPs group(P<0.05).Compared with the Ag NPs group,the frequency of head swing and body bending of the C.elegans increased significantly at 50μg/m L in the rh-MT-Ⅲαplus the Ag NPs treatment groups,indicating that rh-MT-Ⅲαhas a recovery on the reduction in the movement behavior of C.elegans induced by Ag NPs,and the recovery effect increased following as the increase of rh-MT-Ⅲαdose.The head swing frequency of C.elegans returned to normal levels at a dose of 50μg/m L,and the body bending frequency returned to normal levels at a dose of 500μg/m L.(2)The results of growth showed that,compared with the control group,the length and width of C.elegans decreased in Ag NPs group.Compared with Ag NPs PVP group,the length and width of C.elegans increased with the increase of rh-MT-Ⅲαdose in Ag NPs solution plus rh-MT-Ⅲαtreatment groups,while the length and width increased significantly at 50μg/m L and the width could recovery to normal level;(3)The results of ROS content showed that,compared with the control group,the ROS content increased in the Ag NPs group.Compared with Ag NPs groups,the ROS content of C.elegans decreased with the increase of rh-MT-Ⅲαdose in the rh-MT-Ⅲαplus the Ag NPs treatment group,and the ROS content of C.elegans reduced significantly at the dose of 50μg/m L.Based on the results of movement,growth and ROS content,it could be preliminarily proved that rh-MT-Ⅲαhad a relief effect on C.elegans toxicity induced by 20 nm Ag NPs.The rh-MT-Ⅲαwas the human metallothionein Ⅲαpeptide obtained by Suzhou Huihan Biotechnology Co.,Ltd.,which was produced by high-density fermentation and purification of escherichia coli-genetic engineering.The results of toxicity evaluation showed that rh-MT-Ⅲαhad no significant toxicity on cell viability,morphology,membrane and apoptosis rate of the two cells,while the results was consistent with the results on C.elegans that rh-MT-Ⅲαhad no significant toxicity on movement,feeding,growth,lifespan and reproductive function of C.elegans at the experimental dose.The functional investigation showed that rh-MT-Ⅲαcould effectively protect the oxidative damage to Ha Ca T cells induced by UVB at dose of100μg/m L.Detection of free radical clearance in vitro and the ROS results suggested that rh-MT-Ⅲαmight reduce the level of oxidative damage to the body,thereby playing a role in anti-oxidative function.The results of the relief effect of rh-MT-Ⅲαon toxicity to C.elegans induced by nanosilver show that rh-MT-Ⅲαcould effectively reduce the toxicity of Ag NPs to C.elegans at dose of 50μg/m L,and could recovery to normal level.This study explored the potential toxic effects of rh-MT-Ⅲαand evaluated its effects including anti-oxidative and relief toxicity induced by Ag NPs in vitro and in vivo,which can provide a valuable reference for the toxicity evaluation and functional application of rh-MT-Ⅲα. |
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