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[1]陳俊,吳廣文,許惠鳳,等.獨(dú)活寄生湯含藥血清對(duì)大鼠退變軟骨細(xì)胞蛋白激酶 R樣內(nèi)質(zhì)網(wǎng)激酶/免疫球蛋白結(jié)合蛋白信號(hào)通路的影響[J].中醫(yī)正骨,2018,30(08):1-10.
　CHEN Jun,WU Guangwen,XU Huifeng,et al.Effect of Duhuo Jisheng Tang(獨(dú)活寄生湯)medicated serum on protein kinase R-like endoplasmic reticulum kinase/immunoglobulin-binding protein signaling pathway in degenerated chondrocytes of rats[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2018,30(08):1-10.
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獨(dú)活寄生湯含藥血清對(duì)大鼠退變軟骨細(xì)胞蛋白激酶 R樣內(nèi)質(zhì)網(wǎng)激酶/免疫球蛋白結(jié)合蛋白信號(hào)通路的影響()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第30卷
期數(shù):
2018年08期
頁碼:
1-10
欄目:
基礎(chǔ)研究
出版日期:
2018-08-20

文章信息/Info

Title:
Effect of Duhuo Jisheng Tang(獨(dú)活寄生湯)medicated serum on protein kinase R-like endoplasmic reticulum kinase/immunoglobulin-binding protein signaling pathway in degenerated chondrocytes of rats
作者:
陳俊1; 吳廣文2; 許惠鳳2; 鄭春松2; 戴一琛1; 邱建清1; 劉淑如1; 陳振沅1; 劉獻(xiàn)祥2
1.福建中醫(yī)藥大學(xué),福建 福州 350122; 2.福建省中西醫(yī)結(jié)合老年性疾病重點(diǎn)實(shí)驗(yàn)室,福建 福州 350122
Author(s):
CHEN Jun1; WU Guangwen2; XU Huifeng2; ZHENG Chunsong2; DAI Yichen1; QIU Jianqing1; LIU Shuru1; CHEN Zhenyuan1; LIU Xianxiang2
1.Fujian University of Traditional Chinese Medicine,Fuzhou 350122,Fujian,China 2.Fujian Key Laboratory of Integrated Medicine on Geriatrics,Fuzhou 350122,Fujian,China
關(guān)鍵詞:
骨關(guān)節(jié)炎;  軟骨細(xì)胞;  獨(dú)活寄生湯;  信號(hào)傳導(dǎo);  大鼠
Keywords:
osteoarthritis;  chondrocytes;  Duhuo Jisheng Tang;  signal transduction;  rats
摘要:
觀察獨(dú)活寄生湯含藥血清對(duì)大鼠退變軟骨細(xì)胞蛋白激酶R樣內(nèi)質(zhì)網(wǎng)激酶(protein kinase R-like endoplasmic reticulum kinase,PERK)/免疫球蛋白結(jié)合蛋白(immunoglobulin-binding protein,BiP)信號(hào)通路的影響。方法:將36只2月齡清潔級(jí)雄性SD大鼠隨機(jī)分為含藥血清組和空白血清組,每組18只。含藥血清組按照9.3 g·kg-1以獨(dú)活寄生湯灌胃、空白血清組按照10 mL·kg-1以生理鹽水灌胃,每天1次。連續(xù)灌胃7 d后,麻醉下行腹主動(dòng)脈采血,獲取獨(dú)活寄生湯含藥血清和空白血清。取4周齡SD大鼠膝關(guān)節(jié),分離軟骨進(jìn)行軟骨細(xì)胞培養(yǎng),傳代至第3代,以白細(xì)胞介素-1β干預(yù)24 h后獲得退變軟骨細(xì)胞。將獲得的退變軟骨細(xì)胞分為2組,分別以獨(dú)活寄生湯含藥血清(獨(dú)活寄生湯組)和空白血清(空白組)進(jìn)行干預(yù)。分別在干預(yù)24 h、48 h和72 h后,測(cè)定2組退變軟骨細(xì)胞凋亡率、PERK mRNA含量、Bip mRNA含量、真核細(xì)胞翻譯起始因子-2α(eukaryotic initiation factor 2α,eIF-2α)mRNA含量、轉(zhuǎn)錄激活因子-4(activating transcription factor 4,ATF-4)mRNA含量、生長(zhǎng)抑制DNA損傷基因153抗原(growth arrest and DNA damage-inducible 153,GADD153)mRNA含量、半胱氨酸天冬氨酸蛋白酶(cysteine containing aspartate specific protease,Caspase)-9活性及Caspase-3活性。結(jié)果:①退變軟骨細(xì)胞凋亡率。時(shí)間因素與分組因素存在交互效應(yīng)(F=9.022,P=0.038)。2組軟骨細(xì)胞凋亡率總體比較,差異有統(tǒng)計(jì)學(xué)意義,即存在分組效應(yīng)(F=325.246,P=0.000); 干預(yù)后不同時(shí)點(diǎn)之間軟骨細(xì)胞凋亡率的差異有統(tǒng)計(jì)學(xué)意義,即存在時(shí)間效應(yīng)(F=389.768,P=0.000); 2組軟骨細(xì)胞凋亡率隨時(shí)間變化均呈降低趨勢(shì); 干預(yù)后各時(shí)點(diǎn)獨(dú)活寄生湯組軟骨細(xì)胞凋亡率均低于空白組(t=14.055,P=0.000; t=13.264,P=0.000; t=12.183,P=0.000)。②退變軟骨細(xì)胞中PERK mRNA含量。時(shí)間因素與分組因素不存在交互效應(yīng)(F=3.002,P=0.157); 獨(dú)活寄生湯組軟骨細(xì)胞中PERK mRNA含量總體低于空白組,即存在分組效應(yīng)(F=17.833,P=0.013); 干預(yù)后不同時(shí)點(diǎn)之間軟骨細(xì)胞中PERK mRNA含量的差異有統(tǒng)計(jì)學(xué)意義,即存在時(shí)間效應(yīng)(F=43.060,P=0.003); 2組軟骨細(xì)胞中PERK mRNA含量隨時(shí)間變化均呈降低趨勢(shì)。③退變軟骨細(xì)胞中Bip mRNA含量。時(shí)間因素與分組因素不存在交互效應(yīng)(F=4.092,P=0.106); 獨(dú)活寄生湯組軟骨細(xì)胞中Bip mRNA含量總體低于空白組,即存在分組效應(yīng)(F=15.136,P=0.018); 干預(yù)后不同時(shí)點(diǎn)之間軟骨細(xì)胞中Bip mRNA含量的差異有統(tǒng)計(jì)學(xué)意義,即存在時(shí)間效應(yīng)(F=62.968,P=0.001); 2組軟骨細(xì)胞中Bip mRNA含量隨時(shí)間變化均呈降低趨勢(shì)。④退變軟骨細(xì)胞中eIF-2α mRNA含量。時(shí)間因素與分組因素不存在交互效應(yīng)(F=2.138,P=0.201); 獨(dú)活寄生湯組軟骨細(xì)胞中eIF-2α mRNA含量總體低于空白組,即存在分組效應(yīng)(F=155.852,P=0.000); 干預(yù)后不同時(shí)點(diǎn)之間軟骨細(xì)胞中eIF-2α mRNA含量的差異有統(tǒng)計(jì)學(xué)意義,即存在時(shí)間效應(yīng)(F=50.328,P=0.000); 2組軟骨細(xì)胞中eIF-2α mRNA含量隨時(shí)間變化均呈降低趨勢(shì),且降低趨勢(shì)基本一致。⑤退變軟骨細(xì)胞中ATF-4 mRNA含量。時(shí)間因素與分組因素存在交互效應(yīng)(F=14.612,P=0.017); 2組軟骨細(xì)胞中ATF-4 mRNA含量總體比較,組間差異有統(tǒng)計(jì)學(xué)意義,即存在分組效應(yīng)(F=33.703,P=0.004); 干預(yù)后不同時(shí)點(diǎn)之間軟骨細(xì)胞中ATF-4 mRNA含量的差異有統(tǒng)計(jì)學(xué)意義,即存在時(shí)間效應(yīng)(F=187.700,P=0.000); 2組軟骨細(xì)胞中ATF-4 mRNA含量隨時(shí)間變化均呈降低趨勢(shì); 干預(yù)后各時(shí)點(diǎn)獨(dú)活寄生湯組軟骨細(xì)胞中ATF-4 mRNA含量均低于空白組(t=4.343,P=0.012; t=3.915,P=0.017; t=10.719,P=0.000)。⑥退變軟骨細(xì)胞中GADD153 mRNA含量。時(shí)間因素與分組因素不存在交互效應(yīng)(F=3.053,P=0.116); 獨(dú)活寄生湯組軟骨細(xì)胞中GADD153 mRNA含量總體低于空白組,即存在分組效應(yīng)(F=16.946,P=0.015); 干預(yù)后不同時(shí)點(diǎn)之間軟骨細(xì)胞中GADD153 mRNA含量的差異有統(tǒng)計(jì)學(xué)意義,即存在時(shí)間效應(yīng)(F=88.206,P=0.000); 2組軟骨細(xì)胞中GADD153 mRNA含量隨時(shí)間變化均呈降低趨勢(shì)。⑦退變軟骨細(xì)胞中Caspase-9活性。時(shí)間因素與分組因素不存在交互效應(yīng)(F=1.266,P=0.327); 獨(dú)活寄生湯組軟骨細(xì)胞中Caspase-9活性總體低于空白組,即存在分組效應(yīng)(F=21.164,P=0.010); 干預(yù)后不同時(shí)間點(diǎn)之間軟骨細(xì)胞中Caspase-9活性的差異有統(tǒng)計(jì)學(xué)意義,即存在時(shí)間效應(yīng)263.384,P=0.000); 2組軟骨細(xì)胞中Caspase-9活性隨時(shí)間變化均呈降低趨勢(shì),且降低趨勢(shì)基本一致。⑧退變軟骨細(xì)胞中Caspase-3活性。時(shí)間因素與分組因素不存在交互效應(yīng)(F=1.346,P=0.314); 獨(dú)活寄生湯組軟骨細(xì)胞中Caspase-3活性總體低于空白組,即存在分組效應(yīng)(F=19.422,P=0.012); 干預(yù)后不同時(shí)間點(diǎn)之間軟骨細(xì)胞中Caspase-3活性的差異有統(tǒng)計(jì)學(xué)意義,即存在時(shí)間效應(yīng)(F=130.649,P=0.000); 2組軟骨細(xì)胞中Caspase-3活性隨時(shí)間變化均呈降低趨勢(shì),且降低趨勢(shì)基本一致。結(jié)論:獨(dú)活寄生湯含藥血清可通過調(diào)控大鼠退變軟骨細(xì)胞PERK/Bip信號(hào)通路,抑制因內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)引起的軟骨細(xì)胞凋亡。
Abstract:
To observe the effect of Duhuo Jisheng Tang(獨(dú)活寄生湯,DHJST)medicated serum on protein kinase R-like endoplasmic reticulum kinase(PERK)/immunoglobulin-binding protein(BiP)signaling pathway in degenerated chondrocytes of rats.Methods:Thirty-six 2-month-old clean-grade male SD rats were randomly divided into medicated serum group and blank serum group,18 rats in each group.The rats in medicated serum group were intragastric administrated with DHJST in dosage of 9.3 g/kg,while the others in blank serum group were intragastric administrated with the normal saline in dosage of 10 mL/kg,once a day for consecutive 7 days.After the last intragastric administration,their blood were fetched out from abdominal aorta under anesthesia and were made into DHJST medicated serum and blank serum respectively.The knee joints of 4-week-old SD rats were fetched out and the chondrocytes were isolated from knee articular cartilage and were cultured.The third-generation chondrocytes were intervented by interleukin-1β(IL-1β)for 24 hours and then the degenerated chondrocytes were obtained.The obtained degenerated chondrocytes were divided into 2 groups and were intervened by DHJST medicated serum(DHJST group)and blank serum(blank group)respectively.The degenerated chondrocytes apoptosis rate,the contents of PERK mRNA,Bip mRNA,eukaryotic initiation factor 2α(eIF-2α)mRNA,activating transcription factor 4(ATF-4)mRNA and growth arrest and DNA damage-inducible 153(GADD153)mRNA and the activities of cysteine containing aspartate specific protease(Caspase)-9 and Caspase-3 in degenerated chondrocytes were detected at 24,48 and 72 hours after the intervention respectively.Results:There was interaction between time factor and group factor in degenerated chondrocytes apoptosis rate(F=9.022,P=0.038).There was statistical difference in degenerated chondrocytes apoptosis rate between the 2 groups in general,in other words,there was group effect(F=325.246,P=0.000).There was statistical difference in chondrocytes apoptosis rate between different timepoints after the intervention,in other words,there was time effect(F=389.768,P=0.000).The chondrocytes apoptosis rate presented a time-dependent decreasing trend in both of the 2 groups.The chondrocytes apoptosis rate was lower in DHJST group compared to blank group at different timepoints after intervention(t=14.055,P=0.000; t=13.264,P=0.000; t=12.183,P=0.000).There was no interaction between time factor and group factor in the content of PERK mRNA in degenerated chondrocytes(F=3.002,P=0.157).The content of PERK mRNA in chondrocytes was lower in DHJST group compared to blank group in general,in other words,there was group effect(F=17.833,P=0.013).There was statistical difference in content of PERK mRNA in chondrocytes between different timepoints after intervention,in other words,there was time effect(F=43.060,P=0.003).The content of PERK mRNA in chondrocytes presented a time-dependent decreasing trend in both of the 2 groups.There was no interaction between time factor and group factor in the content of Bip mRNA in degenerated chondrocytes(F=4.092,P=0.106).The content of Bip mRNA in chondrocytes was lower in DHJST group compared to blank group in general,in other words,there was group effect(F=15.136,P=0.018).There was statistical difference in content of Bip mRNA in chondrocytes between different timepoints after intervention,in other words,there was time effect(F=62.968,P=0.001).The content of Bip mRNA in chondrocytes presented a time-dependent decreasing trend in both of the 2 groups.There was no interaction between time factor and group factor in the content of eIF-2α mRNA in degenerated chondrocytes(F=2.138,P=0.201).The content of eIF-2α mRNA in chondrocytes was lower in DHJST group compared to blank group in general,in other words,there was group effect(F=155.852,P=0.000).There was statistical difference in content of eIF-2α mRNA in chondrocytes between different timepoints after intervention,in other words,there was time effect(F=50.328,P=0.000).The content of eIF-2α mRNA in chondrocytes presented a time-dependent decreasing trend in both of the 2 groups,and the 2 groups were consistent with each other in the decreasing trend of content of eIF-2α mRNA in chondrocytes.There was interaction between time factor and group factor in the content of ATF-4 mRNA in degenerated chondrocytes(F=14.612,P=0.017).There was statistical difference in content of ATF-4 mRNA in chondrocytes between the 2 groups in general,in other words,there was group effect(F=33.703,P=0.004).There was statistical difference in content of ATF-4 mRNA in chondrocytes between different timepoints after intervention,in other words,there was time effect(F=187.700,P=0.000).The content of ATF-4 mRNA in chondrocytes presented a time-dependent decreasing trend in both of the 2 groups.The content of ATF-4 mRNA in chondrocytes was lower in DHJST group compared to blank group at different timepoints after intervention(t=4.343,P=0.012; t=3.915,P=0.017; t=10.719,P=0.000).There was no interaction between time factor and group factor in content of GADD153 mRNA in degenerated chondrocytes(F=3.053,P=0.116).The content of GADD153 mRNA in chondrocytes was lower in DHJST group compared to blank group in general,in other words,there was group effect(F=16.946,P=0.015).There was statistical difference in content of GADD153 mRNA in chondrocytes between different timepoints after intervention,in other words,there was time effect(F=88.206,P=0.000).The content of GADD153 mRNA in chondrocytes presented a time-dependent decreasing trend in both of the 2 groups.There was no interaction between time factor and group factor in the activity of Caspase-9 in degenerated chondrocytes(F=1.266,P=0.327).The activity of Caspase-9 in chondrocytes was lower in DHJST group compared to blank group in general,in other words,there was group effect(F=21.164,P=0.010).There was statistical difference in activity of Caspase-9 in chondrocytes between different timepoints after intervention,in other words,there was time effect(F=263.384,P=0.000).The activity of Caspase-9 in chondrocytes presented a time-dependent decreasing trend in both of the 2 groups,and the 2 groups were consistent with each other in the decreasing trend of activity of Caspase-9 in chondrocytes.There was no interaction between time factor and group factor in activity of Caspase-3 in degenerated chondrocytes(F=1.346,P=0.314).The activity of Caspase-3 in chondrocytes was lower in DHJST group compared to blank group in general,in other words,there was group effect(F=19.422,P=0.012).There was statistical difference in activity of Caspase-3 in chondrocytes between different timepoints after intervention,in other words,there was time effect(F=130.649,P=0.000).The activity of Caspase-3 in chondrocytes presented a time-dependent decreasing trend in both of the 2 groups,and the 2 groups were consistent with each other in the decreasing trend of activity of Caspase-3 in chondrocytes.Conclusion:DHJST medicated serum can inhibit the chondrocyte apoptosis caused by endoplasmic reticulum stress reaction through regulating PERK/BiP signaling pathway in the degenerated chondrocytes of rats.

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備注/Memo

備注/Memo:
基金項(xiàng)目:國(guó)家自然科學(xué)基金項(xiàng)目(81573801); 福建省自然科學(xué)基金杰青項(xiàng)目(2017J06018); 福建省衛(wèi)生計(jì)生科研人才培養(yǎng)項(xiàng)目(2017-ZQN-62) 通訊作者:劉獻(xiàn)祥 E-mail:[email protected]
更新日期/Last Update: 2018-08-31