• Journal of Oral Medicine and Pain
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• v.31 no.1
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• pp.79-89
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• 2006

The aim of this study is to know how the rat submandibular gland changes under various emotional stress condition, using molecular biological methods. Restraint and chronic unpredictable mild stress (CUMS) experiment is conducted on fifty one 7-week old Sprague-Dawley rats (restraint stress experiment: 21, CUMS: 30). The rats were sacrificed, the submandibular glands were excised immediately at certain time, and examined by the use of immunohistochemistry and western blotting. In CUMS experiment, sucrose preference test, water intake change, weight change were implemented at 1 week interval for the experimental period The results are as follows: 1. The number of clusterin-secreting cells of restraint stress group compared to control group showed significantly decreasing tendency in all experimental groups except for the 1st hour group (p<0.001 in the 9th, 24th, 72nd, 120th, and 168th hour group). 2. The number of clusterin-secreting cells of CUMS group compared to control group showed significantly increasing tendency in the 2nd week group (p<0.01), and significantly decreasing tendency in the 4th and 5th week group (p<0.001). 3. Sucrose preference test in CUMS experiment showed significant difference between the 5th week experimental group and control group (p<0.01). 4. Weight change in CUMS experiment showed significant difference between the 5th week experimental group and control group (p<0.01), but water intake change didn't show significant difference compared to control group. 5. In western blot analysis, clusterin expression was decreased on a gradual basis in due time compared to the control group in the restraint stress group. As for CUMS group (chronic unpredictable mild stress group), it was increased till the 2nd week and decreased till the 5th week after that, which is similar to immunohistochemical analysis result and the decreasing tendency of sucrose preference and weigh changes. Through the test, it was proved that expression of clusterin in saliva glands decreases after receiving either acute or chronic stress, indicating relation with depression caused by chronic stress. Unlike other data, however, apoptotic tendency was hardly found in tissues. Diverse possibilities could be suggested on that: first, the stress was not enough to expedite apoptosis; second, apoptosis-related protein was already being secreted though not detected with microscope; third, clusterin, a major secretion molecule of saliva, decreased with saliva's malfunction due to stress. In the respect, it will be necessary to examine proteins expressed in case of cell death or other heat-shock proteins at the same time, in order to see whether any cellular change or death is caused by decreasing clusterin under high stress, and whether the original state is restored as time goes by under mild stress, through longer-term tests using even higher acute stress.

• Journal of Life Science
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• v.32 no.1
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• pp.11-22
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• 2022

N-methyl-D-aspartate (NMDA) receptors have received considerable attention regarding their involvement in glutamate-induced neuronal excitotoxicity. Resveratrol has been shown to exhibit neuroprotective effects against this kind of overactivation, but the underlying cellular mechanisms are not yet clearly understood. In this study, HT-22 neuronal cells were treated with NMDA in Mg²⁺-free buffer and subsequently used as an experimental model of glutamate excitotoxicity to elucidate the mechanisms of resveratrol-induced neuroprotection. We found that NMDA treatment causes a drop in MTT reduction ability, disrupts inside-negative transmembrane potential of mitochondria, depletes cellular ATP levels, and stimulates intracellular ROS production. Double fluorescence imaging studies demonstrated an increased formation of mitochondrial permeability transition (MPT) pores accompanied by apoptotic cell death, while cobalt protoporphyrin and bilirubin showed protective effects against NMDA-induced mitochondrial injury. On the other hand, zinc protoporphyrin IX significantly attenuated the protective effects of resveratrol which was itself shown to enhance heme oxygenase-1 (HO-1) mRNA and protein expression levels. In cells transfected with HO-1 small interfering RNA, resveratrol failed to suppress the NMDA-induced effects on MTT reduction ability and MPT pore formation. The present study suggests that resveratrol may prevent mitochondrial injury in NMDA- treated HT-22 cells and that enhanced expression of HO-1 is involved in the underlying cellular mechanism.

• Journal of Embryo Transfer
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• v.29 no.1
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• pp.73-81
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• 2014

The in vitro production of porcine embryos was essential to increase of blastocyst development rate and select of high quality blastocyst in early stage. There were a lot of reports about in vitro porcine embryo development, but there was no report about the selection of high quality embryos. Therefore, in this study, we investigated the effect of vitamin $K_1$ (vit $K_1$) on the development and survival rate of porcine in vitro fertilized embryos. When vit $K_1$ was treated for 24 hr at day 1 in vitro culture, blastocyst development rate in the control group ($35.5{\pm}3.2%$) was significantly lower compared to $1.0{\mu}M$, $3.0{\mu}M$, or $6.0{\mu}M$ groups ($14.5{\pm}4.3$, 0.0, or 0.0%; p<0.05). The survival rates of blastocysts at day 8 in $1.0{\mu}M$, $3.0{\mu}M$ or $6.0{\mu}M$ of vit $K_1$ treated groups ($22.2{\pm}2.9$, 0.0 or 0.0%) were significantly lower than that of the control group ($31.8{\pm}2.6%$; p<0.05). We were added at $1.0{\mu}M$, $3.0{\mu}M$ or $6.0{\mu}M$ vit $K_1$ for different durations of time at day 1 in vitro culture. The development rate and survival rate in the group of $1.0{\mu}M$ vit $K_1$ for 6 hr was $26.5{\pm}2.9%$ and $47.2{\pm}2.8%$, respectively, which were differed significantly in the group of 12 hr (p<0.05). In the group of $3.0{\mu}M$ vit $K_1$, the blastocyst development in control group was $36.4{\pm}3.1%$ but, the survival rate $41.7{\pm}3.2%$ in the group of 3.0 hr was significantly higher than that of the control group (p<0.05). In the group of $6.0{\mu}M$ vit $K_1$, the control group's the blastocyst development was $32.0{\pm}2.8%$ and the 0.5 hr supplement group's survival rates was $42.9{\pm}1.8%$ higher than other groups. We added vit $K_1$ at day 1, day 2, day 4 and day 6 of in vitro culture, on the based the results of supplemented concentration and duration. In the group of $1.0{\mu}M$ 6.0 hr addition, the blastocyst development rate of day 4 and the survival rate of day 2 were the highest in each group. In the groups of $3.0{\mu}M$ 3.0 hr addition or $6.0{\mu}M$ 0.5 hr addition, the blastocyst development ($59.5{\pm}4.1%$ and $50.0{\pm}3.6%$) and survival rates ($72.7{\pm}5.4%$ and $79.2{\pm}4.0%$) on day 4 were significantly higher than that of control and other experiment groups (p<0.05). Meanwhile, the number of cells in blastocysts that produced by vit $K_1$ supplementation was $53.4{\pm}5.8$, $49.4{\pm}3.8$ and $51.5{\pm}4.5$ respectively, which were significantly higher than that of $40.2{\pm}2.3$ in the control group (p<0.05). There was no difference of the number of apoptotic cells between control and experiment groups. In addition, gene expression of survival blastocyst, the Bax mRNA expression was similar between the control and the experiment groups. However, Bcl-xL mRNA expression's in the group of $6.0{\mu}M$ 0.5 hr on day 4 was highest among control and experiment groups (p<0.05). In this study suggested that the control of concentration, duration and time was effective on the survival and cell number of porcine blastocyst derived from in vitro. We are not know what the exact reasons of the effect of vit $K_1$ on embryo development and need to fur ther study. However, vit $K_1$ might be using the selection of high quality porcine blastocyst.