Effects of Boron Supplementation on Expression of Hsp70 in the Spleen of African Ostrich.

Increased synthesis of heat shock protein 70 (Hsp70) occurs in prokaryotes and eukaryotes in response to physiological, environmental, and chemical exposures, thus allowing the cell survival from fatal conditions. Hsp70 cytoprotective properties may be clarified by its anti-apoptotic function. Boron has been reported to play an essential role in various organ developments and metabolisms. However, it is not known if boron is also able to modulate the Hsp70. In the present study, the actions of boron on ostrich spleen and expression level of Hsp70 were investigated. Thirty healthy ostrich chicks were randomly assigned to six groups: groups I, II, III, IV, V, and VI and fed the basal diet spiked with 0-, 40-, 80-, 160-, 320-, and 640-mg boric acid (BA)/L, respectively, in drinking water. The histomorphological examination in the spleen was done by hematoxylin and eosin (HE) staining. The expression level of Hsp70 was analyzed by immunohistochemistry (IHC) and western blotting, and mRNA expression of Hsp70 was investigated by quantitative real-time PCR (qPCR). In order to investigate apoptosis, TUNEL assay reaction in all treatment groups was analyzed. Our results showed that the histological structure of spleen up to 160 mg/L BA supplementation groups well developed. The Hsp70 expression level first induced at low-dose groups (up to group IV) and then inhibited dramatically in high-dose groups (V and VI) while comparing with the group I (0 mg BA). The TUNEL assay reaction revealed that the cell apoptosis amount was decreased in group IV, but in group V and especially in group VI, it was significantly increased (P < 0.01). Taken altogether, proper dietary boron treatment might stimulate ostrich chick spleen development by promoting the Hsp70 expression level and inhibiting apoptosis, while a high amount of boron supplementation would impair the ostrich spleen structure by inhibiting Hsp70 expression level and promoting cell apoptosis.

Effect of Boric Acid Supplementation on the Expression of BDNF in African Ostrich Chick Brain.

The degree of brain development can be expressed by the levels of brain brain-derived neurotrophic factor (BDNF). BDNF plays an irreplaceable role in the process of neuronal development, protection, and restoration. The aim of the present study was to evaluate the effects of boric acid supplementation in water on the ostrich chick neuronal development. One-day-old healthy animals were supplemented with boron in drinking water at various concentrations, and the potential effects of boric acid on brain development were tested by a series of experiments. The histological changes in brain were observed by hematoxylin and eosin (HE) staining and Nissl staining. Expression of BDNF was analyzed by immunohistochemistry, quantitative real-time PCR (QRT-PCR), and enzyme linked immunosorbent assay (ELISA). Apoptosis was evaluated with Dutp-biotin nick end labeling (TUNEL) reaction, and caspase-3 was detected with QRT-PCR. The results were as follows: (1) under the light microscope, the neuron structure was well developed with abundance of neurites and intact cell morphology when animals were fed with less than 160 mg/L of boric acid (groups II, III, IV). Adversely, when boric acid doses were higher than 320 mg/L(groups V, VI), the high-dose boric acid neuron structure was damaged with less neurites, particularly at 640 mg/L; (2) the quantity of BDNF expression in groups II, III, and IV was increased while it was decreased in groups V and VI when compared with that in group I; (3) TUNEL reaction and the caspase-3 mRNA level showed that the amount of cell apoptosis in group II, group III, and group IV were decreased, but increased in group V and group VI significantly. These results indicated that appropriate supplementation of boric acid, especially at 160 mg/L, could promote ostrich chicks' brain development by promoting the BDNF expression and reducing cell apoptosis. Conversely, high dose of boric acid particularly in 640 mg/L would damage the neuron structure of ostrich chick brain by inhibiting the BDNF expression and increasing cell apoptosis. Taken together, the 160 mg/L boric acid supplementation may be the optimal dose for the brain development of ostrich chicks.

Increased Thymic Cell Turnover under Boron Stress May Bypass TLR3/4 Pathway in African Ostrich.

Previous studies revealed that thymus is a targeted immune organ in malnutrition, and high-boron stress is harmful for immune organs. African ostrich is the living fossil of ancient birds and the food animals in modern life. There is no report about the effect of boron intake on thymus of ostrich. The purpose of present study was to evaluate the effect of excessive boron stress on ostrich thymus and the potential role of TLR3/4 signals in this process. Histological analysis demonstrated that long-term boron stress (640 mg/L for 90 days) did not disrupt ostrich thymic structure during postnatal development. However, the numbers of apoptotic cells showed an increased tendency, and the expression of autophagy and proliferation markers increased significantly in ostrich thymus after boron treatment. Next, we examined the expression of TLR3 and TLR4 with their downstream molecular in thymus under boron stress. Since ostrich genome was not available when we started the research, we first cloned ostrich TLR3 TLR4 cDNA from thymus. Ostrich TLR4 was close to white-throated Tinamou. Whole avian TLR4 codons were under purify selection during evolution, whereas 80 codons were under positive selection. TLR3 and TLR4 were expressed in ostrich thymus and bursa of fabricius as was revealed by quantitative real-time PCR (qRT-PCR). TLR4 expression increased with age but significantly decreased after boron treatment, whereas TLR3 expression showed the similar tendency. Their downstream molecular factors (IRF1, JNK, ERK, p38, IL-6 and IFN) did not change significantly in thymus, except that p100 was significantly increased under boron stress when analyzed by qRT-PCR or western blot. Taken together, these results suggest that ostrich thymus developed resistance against long-term excessive boron stress, possibly by accelerating intrathymic cell death and proliferation, which may bypass the TLR3/4 pathway. In addition, attenuated TLRs activity may explain the reduced inflammatory response to pathogens under boron stress.