| In order to reveal the regulatory mechanisms of MT (metallothionein) which a highly effective antioxidant on apoptosis/necrosis in lactating cows under heat stress, the spleen lymphocytes from neonatal male calf were cultured firstly in37℃or43℃for1hour respectively to get varying degrees of apoptosis and necrosis, and adding different working concentration of MT in culture media, by flow cytometry and western-blot. And then,20Holstein dairy cattle in heat stress were divided into4groups:A (0mg/capita), B (4mg/capita), C (8mg/capita) and D (16mg/capita) by intravenous, were conducted with experiment for blood sampling by the law of diminishing time(the first bleed before intravenous injecting). The samples were determined by flow cytometry, western-blot and real-time PCR. It will provide scientific basis and new ways for applying of physiological activator and studying theoretical apoptosis. The results are as follows:1Heat stress increased cell apoptosis. MT only has inhibitory effect on the apoptosis induced by heat stress, while the inhibitory effect of MT on normal apoptosis is not obvious. The necrocytosis can be promoted by the lower MT when the concentration was2.25ug/ml; High fever can protect mitochondria. And the△Ψm value can be increased more by MT which strengthen cellular tissue because of its anti-apoptosis ability, but the high stress of cell require higher concentration of MT (8.25μg/mL), and the nomal cell require moderate concentration, cell can not be protected enough by too high(MT was8.25ug/ml) or too low (MT was2.25ug/ml) concentration of MT. Fever lead to increase the expression of phospho-p50and inhibit the expression of phospho-p65. While the primary spleen lymphocytes cultured in37℃can be increased expression of phospho-p65who mean to main effectiveness of NF-κB. Therefore, because of thermal oxidative stress resulting in decreasing activity of NF-κB, heat stress signal can not be quickly transferred into the nucleus. Heat stress injury caused by apoptosis could be regulated by MT increasing the expression of phospho-P53in case of its protective function. When the working concetration of MT was2.25μg/mL, the production of AP-1could be improved because of the expression of phospho-c-jun in cells in thermal oxidationstress decreased. Thus the production of AP-1could be inhibited by thermal oxidationstress. Therefore, fever improve mitochondrial membrane potential of bovine spleen lymphocytes, and it also led to increasing necrosis, while adding MT in culture media, not only mitochondrial membrane in cell could be improved, but also the necrosis being resulted in heat stress be inhibited better.It has more practical meaning for adopting adding MT to improve the integrity of cellular mitochondria than high fever, at the same time, it also showed that one of signal transduction pathway which MT can regulate apoptosis, necrosis induced heat stress is mitochondrial pathway.2Effects of exogenous metallothionein (MT) on lymphocytes apoptosis, necrosis and mitochondrial transmembrane potential(△Ψm) of China Holstein cows were studied using20lactating cows who being in heat stress, randomly allocated to groups A, B, C and D. MT was supplemented at0.0,4.0,8.0and16.0mg/capita, respectively, by intravenous injection. The results show that corrected milk yields in groups B, C, D were significant higher than those in group A after being injected MT during51-60d(P<0.05); at35d, the lymphocytes apoptosis rate of group C (8mg/capita) significantly lower than those of groups B and D (P<0.05), the lym Corrected milk yields e in group C (8mg/capita) was decreased significantly at35d and50d (P<0.05); And the necrosis rates in group D (16mg/head) in50,60d were significantly lower than in1d, the mitochondrial membrane potential (△Ψ) in group A reach to the supreme at35d, but the△Ψ in group B was significantly rising in35d (P<0.05), the△Ψ in group A and B was not only gone up, but decreased in50d, the△Ψ in group C was constantly during, in group D (16mg/head) kept rising during1～50d and kept constantly during51-60d, the dose of MT was higher resulted in the△Ψ being higher in60d.In conclusion, the content of NO in group C was increased significantly (P<0.05) in35d or50d and extremely significant (P<0.01) in60d comparing to before cows being injected MT.8mg/capitaof MT appeared the greatest impact for the NO in cell. And the content of NO in cell was regulated by necrosis, but the versa was not. At the same time there were35days for inhibiting heat stress leading to injury in cows for injection MT4mg/capita, and there were50days for8mg/capita,60days for16mg/capita. In conclusion, not only the heath of cows being in heat stress can be improved, but also the animals can be converted heat stress to heat stress damage repaired state by the exogenous MT.3The relative expression of HSP70in group C was the highest, and it mean that MT can protect animal in heat stress through promoting the expression of HSP70; HSP90-α expression was increased in35d(except for group B), during50-60d when the animals were at the state of recovering after heat stress injury, HSP90-a expression was rising in experimental groups and falling in control group; in group C, the gene expression of Bcl-2was creased significantly (P <0.05) during35-50d, and the gene expression of Bax was decreased in50d; in group C, the gene expression of Bcl-2was higher significantly in50d than in the other time(P<0.05), and the gene expression of Bcl-2was supressed for injecting16mg/capita; both the gene expression of Bax and Bcl-2were increased in35d, and the gene expression of Bax was decreased while that of Bcl-2was increased in50d which was the moment of recovering from heat stress injury; in the case of these, the gene expression of Bax was inhibited by the gene expression of Bcl-2being increased, that confirmed MT adjusted apoptosis through the synergistic effect of Bcl-2and Bax. In control group, the correlation coefficient between HSP90-α and Bax, Bax and Bcl-2, HSP90-α and Bax were decrease in turn, and there was no significant correlation between HSP70, Bcl-2and Bax; in conclusion, MT increased the gene expression of anti-apoptotic such as bcl-2which lead to strengthen the viability of cells, and regulated the expression of the heat shock in feedback.4The gene expression of c-myc were rising during50-60d in control group, and was higher significantly than in experimental groups (P<0.05); the relative gene expression of p27kipl in group B maintained at from0.66to1.09times during20-50d as before injecting MT, and kept a low level of gene expression; and the relative gene expression of p27kipl in group C was remained stable; the relative gene expression of p27kipl in group D maintained at from1.49to3.05times during20-60d as before injecting MT; there is a high level relative expression of p27kiplduring20-35d in control group; there was a strong positive linear correlation between c-jun and c-fos, Phospho-c-jun and phospho-c-fos in group C, and the linear slope reached to the maximum, it suggested that the activity of AP-1in cells reached to the maximum, and promoted the expression of downstream genes, furthermore, the range of variation on gene expression also reached the maximum corresponding; the gene expression of NF-κB p50in group C was greater significantly than in the other groups in20,50-60d(P<0.05), along with the more time from injection MT, the gene expression of NF-κ B p50kept decreasing, while that of in group A appeared rising during the experiment; therefore, when the injected dose MT was8mg/capita, the gene expression of NF-κ B p50could be activated; when the injected dose MT was4mg/capita, NF-κ B subunit p65kept being inhibited about35days; and when the injected dose MT was8mg/capita,NF-κ B subunit p65kept being inhibited about50days; and when the injected dose MT was16mg/capita, the time of inhibition on NF-κ B subunit p65were more than60days; in conclusion, the greater the dose of MT, the longer time of inhibition on gene expression of p65. the law on MT regulating gene of NF-κ B p50was "all or none", beause of showing the promotion expression in MT dose to8mg/capita, while the chemical dose affection was appeared in the MT regulating the gene expression of p65, and the two laws consist of the regulatory mechanisms of MT on NF-κ B. By controlling the activity and contents of AP-1respectively through regulating the expression of c-jun and c-fos, MT completed the bidirectional regulation on cell proliferation, growth and apoptosis and other physiological activities. |
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