Effects And Mechanisms Of Se-enriched Ligustrum Lucidum Fruit Preventing And Curing On Heat-stressed Mice Study

When animals are subjected to over-high temperature stimulation that is beyond thermo regulation capability of their bodies, systematic indications including systemic indications and the non-specific and specific defense reaction will happen and this phenomenon is called heat stress.To scientifically study the mechanisms of heat stress in animals, many researchers observed the animal test model of blood biochemical indexes, hormone levels, cytokines (e.g. IL-2, IL-6, TNF, etc.) and tissue pathological changes and so on, through establishing heat stress experimental animals, such as chicken, swine, rats and mice. In addition, they also found that under the thermal stress conditions, corresponding changes of heat shock proteins (e.g. Hsp70, Hsp60, Hsp90, etc.) occurred, and they considered that the heat shock protein level of the body is one of the animals thermal stress indexes.In this study, the research objective is to improve the heat tolerance of dairy cows. On the basis of building mice heat stress model, the function of ligustrum lucidum fruit, such as tonifying the yin and kidney, liver protection, immune regulation and so on, were chosen as the breakthrough point and the role of Se-enriched ligustrum lucidum fruit in preventing and curing heat stress was investigated through heat stress mice mortality statistics and observed histopathological lesions in the liver, growth index, immune organ index, blood biochemistry, hormone levels and serum IL-2levels. Besides, the synthesis of HSP70and the expression of its mRNA in the liver that as heat stress related indicators, was detected by using the method of ELISA and real-time fluorescence relative quantification assay.The effect of Se-enriched ligustrum lucidum fruit against heat stress in mice was evaluated through the above physiological indicators and to figure out its mechanisms.Test1Effect of Se-enriched ligustrum lucidum fruit in preventing on acute heat stress in miceThe effect of Se-enriched ligustrum lucidum fruit on survival rate of acute heat stress in mice was studied,126healthy ICR male mice were randomly divided into control group(C), heat stress group(HS), common ligustrtrum lucidum fruit group(CLL), high-dose Se-enriched ligustrum lucidum fruit group(HSLL), Mid-dose Se-enriched ligustrum lucidum fruit group (MSLL) and low-dose Se-enriched ligustrum lucidum fruit group(LSLL) groups. Except C group, other test groups were placed under continuous heat stress for2hours in temperature43±1℃, relative humidity50-60%. During acute heat stress period, the clinical symptoms, mortality rate and biopsy pathology slides of liver tissue were observed. The results showed that the mortality rate of mice in Se-enriched ligustrum lucidum fruit groups were obviously reduced, and the mortality rate in0.75g/kg dose was38.1%, it was extremely lower than the HS group(P<0.01), which had the lowest survival rate79.2%, and it was also lower remarkably than the CLL group mortality rate61.9%(P<0.05). Pathological observation found that the damage of mice liver tissue of the LSLL group, such as edema and granular degeneration of liver cell, was obviously alleviated. It suggested that Se-enriched ligustrum lucidum fruit in0.75g/kg had significantly preventive effect on acute heat stress.Test2Effect of Se-enriched ligustrum lucidum fruit on heat-stressed mice growth performance, serum biochemical levels and morphology of liverIn order to investigate the effect of Se-enriched ligustrum lucidum fruit against heat stress in mice and their clinical indexes, such as growth index, blood biochemistry, histopathological lesions in liver tissue were observed.108healthy ICR male mice were randomly divided into control group(C), heat stress group(HS), common ligustrtrum lucidum group(CLL), high-dose Se-enriched ligustrum lucidum group (HSLL), Mid-dose Se-enriched ligustrum lucidum group(MSLL) and low-dose Se-enriched ligustrum lucidum group(LSLL) groups. Except C group, other test groups were placed under continuous heat stress for2hours in temperature40℃±1℃,relative humidity40-50%, and then in T35±4℃, RH45-50%during the daytime, T28±5℃, RH30-40%in the night, continous suppliance of water or ligustrum lucidum fruit water decoction. The results indicated that under high temperature condition, the values of mice weight growth(GH), specific growth rate(SGR) and feed conversion ratio(FCR) in heat stress groups, compared with the C group, reduced in different degrees, however, the values of WG and SGR in the LSLL group were both significantly higher than the HS group(P<0.05). Each treatment group showed an inscending trend in serum ALT, AST activity, but compared with the other groups the activity of serum ALT and AST of the CLL group and the Se-enriched ligustrum lucidum fruit groups(included HSLL, MSLL and LSLL) decreased in different degrees. On the3rd day of heat stress, the concentration of serum TP, ALB and GLO of the LSLL group was respectively significantly higher than the other treatment groups, and was significantly lower than the CLL group(P<0.05). It showed that0.75g/kg dose of Se-enriched ligustrum lucidum fruit water decoction have a better effect on improving the concentration of serum proteins. The experiment results indicated that Se-enriched ligustrum lucidum fruit and common ligustrum lucidum fruit can obviously alleviate the high temperature damage on growing performance and liver tissue. Especially, Se-enriched ligustrum lucidum fruit in0.75g/kg dose can do the better in decreasing the activity of serum ALT and AST, improving the concentration of serum TP, ALB and GLO, thus enhancing the ablity of anti-heat stress on mice.Test3Effect of Se-enriched ligustrum lucidum fruit on heat-stressed mice immune organs index, the dynamic changes of serum IL-2and hormone levelsTo study immunomodulatory effects of Se-enriched ligustrum lucidum fruit on heat stress in mice, on the basis of above mice heat stress model trial, immune organs index, the dynamic changes of serum hormone(ACTH, CORT, T4, T3) and the cytokine IL-2level were detected. The results indicated that immune organs index of the CLL, MSLL and LSLL groups were significantly higher than those of the HS group(P<0.05). In heat stress early period(1～3d), serum ACTH level of the HS group increased significantly(P<0.05), while CLL, MSLL and LSLL groups had no significantly different(P>0.05). And the heat stress groups CORT levels were remarkably higher than those of the C group(P<0.05), the CORT levels of ligustrum lucidum fruit grous were all reduced, in which there was no significantly different between the LSLL group and the C group (P>0.05). The level of T4and T3taked on decline trend as a whole in the HS group(P<0.05). However, T4and T3levels in ligustrum lucidum fruit groups were much higher than those of the HS group(P<0.01). As for IL-2, in early heat stress period(1～3d), IL-2level in HSLL and LSLL groups was significantly higher than that of C and HS groups(P<0.05, P<0.01).The results demonstrated that Se-enriched ligustrum lucidum fruit can enhance immune regulating function and improve the body’s internal environment condition by the endocrine-immune regulation network system acting. Immunomodulatory function of Se-enriched ligustrum lucidum fruit water decoction in0.75g/kg dose can reach the best result.Test4Effect of Se-enriched ligustrum lucidum fruit on the synthesis of liver Hsp70and the expression of its mRNA in heat-stressed miceIn this experiment, the synthesis of liver HSP70and the expression of its mRNA of different period were detected by ELISA and real-time fluorescence relative quantification assay, respectively, to approach the mechanism of Se-enriched ligustrum lucidum fruit against heat stress. The results showed that the synthesis of mice liver HSP70of each experimental group were on the rise during heat stress period, and in early heat stress period(1～3d), the synthesis of liver HSP70of each Se-enriched ligustrun lucidum fruit group were all significantly higher than the HS group(P<0.05). On the7th day of heat stress, the synthesis of HSP70in CLL and HS groups were remarkably higher than that of the C group(P<0.05), but there was no significantly difference between ligustrum lucidum fruit groups and the C group(P>0.05). The HSP70mRNA expression of heat stress groups were reduced at first, and then increased, and the general was on the rise. On the7th day of heat stress, the HSP70mRNA expression of CLL, MSLL and LSLL groups were all significantly lower than the HS group(P<0.05). The above experimental results demonstrated that Se-enriched ligustrum lucidum fruit and common ligustrum lucidum fruit both can obviously support the HSP70mRNA expression and its protein synthesis in early heat stress period, thus make heat-stressed mice quickly adapt the high temperature environment, and alleviate liver damage caused by thermal stress. In general, Se-enriched ligustrum lucidum fruit water decoction can reach the best result in0.75g/kg dose.Conclusion:Se-enriched ligustrum lucidum fruit can reduce acute heat stress mice mortality effectively, and have apparent preventive and therapeutic effects, especially in0.75g/kg dose. Ligustrum lucidum fruit can not only alleviate mice growth inhibition caused by heat stress, but also increas the concentration of serum TP, ALB and GLO and enhance mice immunity function. The function mechanism of ligustrum lucidum fruit is relevant with many factors, including improvement of serum T3and T4levels, adjustment of heat-stress-derived ACTH levels rising excessively, regulation of endocrine-immune network system, support of HSP70mRNA expression and its protein synthesis of mice liver tissue in early heat stress period, alleviation heat-stress-derived damage of liver tissue and maintainance of internal environment.