• 대한한방부인과학회지
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• 제18권1호
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• pp.181-191
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• 2005

Purpose : ${\beta}$-sitosterol is kind of phytosterols or plant which are structurally similar to cholesterol. This study was aimed to investigate the inhibitory effect of the ${\beta}$-sitosterol on the proliferation of human uterine leiomyoma cells and the expression of gene related the mechanism of cell apoptosis. Methods : We counted the number of death cells treated with indicated time of the ${\beta}$-sitosterol and investigated cell death rate by cell count assay. Furthermore, flow cytometry analysis and DNA fragmentation assay were used to dissect between necrosis and apoptosis. and then we observed the differential gene expression by western blot analysis. Results : 1) The inhibitory effect on the growth of uterine leiomyoma cell treated with the ${\beta}$-sitosterol $16{\mu}M$ was increased in a time dependent. 2) The result of flow cytometry analysis, subG1 phase arrest related cell apoptosis was investigated 16.97% in uterine leiomyoma cell treated with the ${\beta}$-sitosterol $16{\mu}M$ and showed the fashion of proportional time dependent. 3) The gene expression of p27, p21 related cell cycle was increased according to increasing time interval but cyclin E-CDK2 complex was decreased expression. 4) The character of apoptosis, DNA fragmentation was significantly observed on the time dependent. 5) The expression of pro-caspase 3 and PARP were decreased dependent on treatment with time dependent. Conclusion : This study showed that the ${\beta}$-sitosterol have the inhibitory effect on the proliferation of human uterine leiomyoma cell and the effect was related with apoptosis.

• Journal of Chest Surgery
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• 제33권12호
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• pp.935-940
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• 2000

배경: 폐혈관 손상에 관한 기전은 여러 보고에도 불구하고 자세히 밝혀지지는 않았다. 최근 산화성 스트레스 질환에 관여하는 과산화 수소($H_2O$$_2$) 등의 활성 산소족(reactive oxygen species)은 세포손상과 세포자사(apoptosis)에 중요한 역할을 한다고 알려져 있다. 본 연구에서는 $H_2O$$_2$에 의하여 유발된 산화성 스트레스가, 폐혈관 손상 기전의 하나로 추측되고 있는 세포자사를 야기하는지를 연구하였다. 대상 및 방법: 소의 폐동맥에서 유래된 calf pupmonary artery endothelial cell line(CPAE)를 이용하였다. $H_2O$$_2$에 의한 세포 독성을 측정하기 위하여, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide(MTT) assay를 시행하였다. $H_2O$$_2$에 의한 세포의 형태학적 변화는 도립 현미경으로 분석하였다. 세포자사를 확인하기 위하여 terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL) assay와 4,6-diamidino-2-phenylindole(DAPI) staining 방법 및 flow cytometry 분석를 시행하였다. 결과: $H_2O$$_2$에 의한 세포 생존율은, 대조군(100%)과 비교하여 3시간 실험군에서 10$\mu$M에서 약 70%, 50 $\mu$M에서 약 33%, 100 $\mu$M에서 약 26%, 500 $\mu$M에서 약 28%이였다. $H_2O$$_2$투여시 세포돌기 감소, 세포 축소, 세포질 응축과 불규칙한 형태 등의 세포자사에 나타나는 형태학적 변화를 나타내었다. TUNEL assay와 DAPI staining에서도 세포자사에 특징적으로 나타나는 핵응축과 핵분절 등의 소견을 나타내었다. Flow cytometry 분석 시에도 $H_2O$$_2$투여시 sub G$_1$분절의 증가와 G$_1$분절의 감소 등의 세포자사 양상이 확인되었다. 결론: 형태학적 분석과 생화학적 분석을 통하여, $H_2O$$_2$는 CPAE에서 세포자사를 야기함을 확인하였다. 이러한 결과는 폐혈관 손상의 기전에 $H_2O$$_2$에 의한 세포자사가 부분적으로 관여할 가능성을 제시한다.

• 식물조직배양학회지
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• 제28권4호
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• pp.179-184
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• 2001

담배 정상주 (Nicotiana tabacum L. cv. BY-4), 약배양으로 유도시킨 반수체 (2n=2x=24), 콜히친 처리로 염색체를 배가한 식물체 (2n=4x=48)및 그 후대식물체 (F$_1$)를 각각 사용하여 배수성에 따른 공변세포 내 엽록체 수를 조사하였다. 공변세포 내 엽록체의 수는 정상주에서 반수체의 2배로 배수성과 밀접한 관계를 나타내었다. 또한, 반수체를 콜히친으로 배가시킨 식물체에서 공변세포 내 엽록체의 수는 반수체의 2배로 배수성이 회복됨에 따라 증가하였다. 이배체의 후대식물에서도 공변세포 내 엽록체의 수는 반수체의 2배로 나타났다. 엽령에 따른 공변세포 내 엽록체 수의 변동도 관찰되지 않았다. 이러한 결과는 기공의 공변세포 내에 분포하는 엽록체 수와 배수성 (2x와 4x)과는 밀접한 관계가 있음을 시사하는 것이다. 기내에서 생장한 식물체에서 공변세포 내 엽록체의 수는 pot 재배식물체와 비슷한 결과를 나타내었다. 따라서 잎의 공변세포 내 기공수는 배수성을 간단하게 판정하는데 이용할 수 있을 것으로 보인다.

• 대한안과학회지
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• 제59권11호
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• pp.1056-1061
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• 2018

목적: 발프로익산이 배양된 인체 테논낭 섬유아세포의 생존에 미치는 영향에 대해 알아보고자 하였다. 대상과 방법: 일차배양한 섬유아세포에 0, 0.25, 0.5, 1.0 mM 발프로익산과 0, 1.0, $2.5{\mu}g/mL$ 미토마이신 C에 각각 또는 동시에 5일간 노출시킨 후 MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay를 이용하여 세포의 생존을 측정하였고, annexin-V/propidium iodide 이중염색 후 유세포 분석을 이용하여 세포고사의 정도를 측정하였다. 결과: 발프로익산은 농도에 비례하여 섬유아세포의 생존을 유의하게 저하시켰으며, 미토마이신 C를 추가한 경우 섬유아세포의 생존을 더욱 저하시켰다. 두 약제 모두 섬유아세포의 세포고사를 유발하였으나 발프로익산은 미토마이신 C에 비해 세포고사를 적게 유발하였다. 결론: 발프로익산은 섬유아세포의 생존을 저하시키며 세포고사를 유발한다. 또한 미토마이신 C를 병용한 경우 섬유아세포에 대한 항증식효과가 더 나타날 것으로 생각된다.

• 고려인삼학회:학술대회논문집
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• 고려인삼학회 2002년도 학술대회지
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• pp.376-384
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• 2002

Object To find out which of the 27 ginsenosides isolated from Panax ginseng C.A. Mey that may inhibit the proliferation of human osteosaocoma cell line $U_2OS$. Methods Effects of each individual ginsenoside on the proliferation of $U_2OS$ cell were studied by determining the viability of cancer cells during culture with or without the presence of the test compound. DNA assay was determined by flow cytometry. Results Ginsonosides -Ro, $-Rh_l,\;-Rh_2,\;-F_1\;and\;-L_8$ at concentrations of 5 ,umol/L could obviously suppress the proliferation of $U_2OS$ cells while ginsenosides $-Rg_1,\;-F_3,$ -Rf, PPT and PT significantly inhibited the cancer cells. Flow cytometry revealed that ginsenosides $-Ro,-Rg_1-Rf,-F_1-Rh_2,PPT$ and PT induced cell cycle arrest at $G_0/G_1$ phase with obvious decrease of cell count at Sand $G_2+M$ phase, Moreover, ginsenosides $-Rf_1,-Rg_1,\;-F_1$ and PPT induced significantly high rates of cell death as compared with the control. Conclusion These data suggested that ginsenosides inhibited $U_2OS$ proliferation Via cell cycle arrest or induction of cell death.

• Journal of Veterinary Science
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• 제22권2호
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• pp.18.1-18.12
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• 2021

Background: We previously elucidated the protective mechanism of Korean red ginseng oil (RGO) against Brucella abortus infection, and our phytochemical analysis revealed that palmitic acid (PA) was an abundant component of RGO. Consequently, we investigated the contribution of PA against B. abortus. Objectives: We aimed to investigate the efficacy of PA against B. abortus infection using a murine cell line and a murine model. Methods: Cell viability, bactericidal, internalization, and intracellular replication, western blot, nitric oxide (NO), and superoxide (O₂^-) analyses and flow cytometry were performed to determine the effects of PA on the progression of B. abortus infection in macrophages. Flow cytometry for cytokine analysis of serum samples and bacterial counts from the spleens were performed to determine the effect of PA in a mouse model. Results: PA did not affect the growth of B. abortus. PA treatment in macrophages did not change B. abortus uptake but it did attenuate the intracellular survivability of B. abortus. Incubation of cells with PA resulted in a modest increase in sirtuin 1 (SIRT1) expression. Compared to control cells, reduced nitrite accumulation, augmented O₂^-, and enhanced pro-inflammatory cytokine production were observed in PA-treated B. abortus-infected cells. Mice orally treated with PA displayed a decreased serum interleukin-10 level and enhanced bacterial resistance. Conclusions: Our results suggest that PA participates in the control of B. abortus within murine macrophages, and the in vivo study results confirm its efficacy against the infection. However, further investigations are encouraged to completely characterize the mechanisms involved in the inhibition of B. abortus infection by fatty acids.

• Biomolecules & Therapeutics
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• 제27권1호
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• pp.48-53
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• 2019

Reactive oxygen species (ROS) are widely generated in biological processes such as normal metabolism and response to xenobiotic exposure. While ROS can be beneficial or harmful to cells and tissues, generation of ROS by diverse anti-cancer drugs or phytochemicals plays an important role in the induction of apoptosis. We recently identified a derivative of naphthalene, MS-5, that induces apoptosis of an ovarian cell, CAOV-3. Interestingly, MS-5 induced apoptosis by down-regulating the ROS. Cell viability was evaluated by water-soluble tetrazolium salt (WST-1) assay. Apoptosis was evaluated by flow cytometry analysis. Intracellular ROS ($H_2O_2$), mitochondrial superoxide, mitochondrial membrane potential (MMP) and effect on cycle were determined by flow cytometry. Protein expression was assessed by western blotting. The level of ATP was measured using ATP Colorimetric/Fluorometric Assay kit. MS-5 inhibited growth of ovarian cancer cell lines, CAOV-3, in a concentration- and time-dependent manner. MS-5 also induced G1 cell cycle arrest in CAOV-3 cells, while MS-5 decreased intracellular ROS generation. In addition, cells treated with MS-5 showed the decrease in MMP and ATP production. In this study, we found that treatment with MS-5 in CAOV-3 cells induced apoptosis but decreased ROS level. We suspect that MS-5 might interfere with the minimum requirements of ROS for survival. These perturbations appear to be concentration-dependent, suggesting that MS-5 may induce apoptosis by interfering with ROS generation. We propose that MS-5 may be a potent therapeutic agent for inducing apoptosis in ovarian cancer cell through regulation of ROS.

• 대한한의학회지
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• 제40권2호
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• pp.35-50
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• 2019

Objectives: Orostachys japonicas (O. japonicus) has been known for its anti-tumor effect. In the present study, it was investigated whether O. japonicus EtOH extracts could induce apoptosis and autophagy which are part of the main mechanism related to anti-tumor effect in THP-1 cells. Methods: Cells were treated with various concentrations of O. japonicus EtOH extracts ($0-300{\mu}g/ml$) for 24, 48, and 72h. Cell viability was evaluated by MTS/PMS assay and apoptosis rate was examined by flow cytometry and ELISA assay. The mRNA expression of apoptosis-related genes (Bcl-2, Mcl-1, Survivin, Bax) and autophagy-related gene (mTOR) was evaluated using real-time PCR. The protein expression of Caspase-3, Akt, LC3 II, Beclin-1, Atg5, $NF-{\kappa}B$, p38, ERK was evaluated using western blot analysis. Results: O. japonicus EtOH extracts inhibited cell proliferation and apoptosis rate was increased in both flow cytometry and ELISA assay. Bcl-2, Mcl-1, Survivin (anti-apoptosis factors) mRNA expressions were decreased and Bax (pro-apoptosis factor) mRNA level was increased. mTOR mRNA expressions was decreased and LC3 II protein expressions was increased. Activation of $NF-{\kappa}B$ was decreased and phosphorylation of p38 was increased. Conclusion: O. japonicus is regarded to inhibit cell proliferation, to induce apoptosis and to regulate autophagy-related genes in THP-1 cells via $NF-{\kappa}B$ and p38 MAPK signaling pathway. This suggests O. japonicus could be an effective herb in treating acute myeloid leukemia.

• Animal Bioscience
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• 제34권8호
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• pp.1321-1330
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• 2021

Objective: Transgenic hens hold a great promise to produce various valuable proteins. Through virus transduction into stage X embryo, the transgene expression under the control of constructed chicken ovalbumin promoters has been successfully achieved. However, a validation system that can evaluate differently developed ovalbumin promoters in in vitro, remains to be developed. Methods: In the present study, chicken oviduct epithelial cells (cOECs) were isolated from oviduct tissue and shortly cultured with keratinocyte complete medium supplemented with chicken serum. The isolated cells were characterized with immunofluorescence, western blot, and flow cytometry using oviduct-specific marker. Chicken mutated ovalbumin promoter (Mut-4.4-kb-pOV) was validated in these cells using luciferase reporter analysis. Results: The isolated cOECs revealed that the oviduct-specific marker, ovalbumin protein, was clearly detected by immunofluorescence, western blot, and flow cytometry analysis revealed that approximately 79.40% of the cells contained this protein. Also, luciferase reporter analysis showed that the constructed Mut-4.4-kb-pOV exhibited 7.1-fold (p<0.001) higher activity in the cOECs. Conclusion: Collectively, these results demonstrate the efficient isolation and characterization of cOECs and validate the activity of the constructed ovalbumin promoter in the cultured cOECs. The in vitro validation of the recombinant promoter activity in cOECs can facilitate the production of efficient transgenic chickens for potential use as bioreactors.

• Clinics in Shoulder and Elbow
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• 제24권4호
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• pp.224-230
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• 2021

Background: Local anesthetics often are used in rotator cuff tears as therapeutic tools, although some cases have reported that they have detrimental effects. Neurotropin (NTP) is used widely in Japan as a treatment for various chronic pain conditions and is shown to have protective effects on cartilage and nerve cells. In this study, we investigated the protective effect of NTP against lidocaine-induced cytotoxicity. Methods: Tenocytes from rotator cuff tendons were incubated with lidocaine, NTP, lidocaine with NTP, and a control medium. Cell viability was evaluated using the WST-8 assay. Cell apoptosis was detected via annexin V staining using flow cytometry. The expression of BCL-2 and cytochrome c, which are involved in the intrinsic mitochondrial pathway of apoptosis, was evaluated via Western blotting and immunohistochemical staining. Results: In the cell viability assay, lidocaine decreased cell viability in a dose-dependent manner, and NTP did not affect cell viability. Moreover, NTP significantly inhibited the cytotoxic effect of lidocaine. The flow cytometry analysis showed that lidocaine significantly induced apoptosis in tenocytes, and NTP considerably inhibited this lidocaine-induced apoptosis. Western blotting experiments showed that lidocaine decreased the protein expression of BCL-2, and that NTP conserved the expression of BCL-2, even when used with lidocaine. Immunohistochemical staining for cytochrome c showed that 0.1% lidocaine increased cytochrome c-positive cells, and NTP suppressed lidocaine-induced cytochrome c expression. Conclusions: NTP suppresses lidocaine-induced apoptosis of tenocytes by inhibiting the mitochondrial apoptotic pathway. Intra-articular/bursal injection of NTP with lidocaine could protect tenocytes in rotator cuff tendons against lidocaine-induced apoptosis.