目的 观察熊果酸对血小板衍生生长因子(PDGF)诱导的大鼠活化型肝星状细胞株(HSC-T6)还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶(NOX)活化及下游信号通路激活的影响.方法 取大鼠HSC-T6,分为空白对照组(不予处理)、熊果酸对照组(50 μmol/L)、PDGF组(10 μg/L)、熊果酸干预组(50 μ mol/L熊果酸+10μg/L PDGF)、二联苯碘干预组(20μmol/L二联苯碘+10 μg/L PDGF)、SB203580干预组(10 μmol/L SB203580+ 10 μg/LPDGF)、LY294002干预组(10 μmol/L LY294002+10 μg/L PDGF)、活性氧阳性对照组(5μg/mL活性氧试剂rosup).采用荧光定量-PCR法检测除活性氧阳性对照组外的其余各组细胞中Ⅰ型胶原mRNA的表达水平.采用Western印迹法检测膜蛋白NOX亚基p47phox(除外活性氧阳性对照组)、磷脂酰肌醇3激酶(PI3K,除外活性氧阳性对照组和SB203580干预组)、磷酸化蛋白激酶B(p-Akt,除外活性氧阳性对照组和SB203580干预组)、磷酸化p38丝裂原活化蛋白激酶(除外活性氧阳性对照组和LY294002干预组)的表达.采用活性氧检测试剂盒和荧光酶标仪检测除SB203580干预组和LY294002干预组外的其余各组细胞内荧光强度.组间比较行单因素方差分析和LSD检验.结果 Ⅰ型胶原mRNA表达水平,PDGF组(3.74±0.32)高于空白对照组(1.00土0.00),熊果酸对照组(0.21±0.02)低于空白对照组,熊果酸干预组(1.02±0.12)、二联苯碘干预组(1.09±0.21)、SB203580干预组(1.18±0.27)、LY294002干预组(1.15±0.26)均低于PDGF组,差异均有统计学意义(t=15.667、-4.501、-15.553、-15.154、-14.642、-14.813,P均＜0.05).p47phox蛋白表达水平,PDGF组(1.98±0.53)高于空白对照组(1.00±0.00),熊果酸对照组(0.48±0.10)低于空白对照组,熊果酸干预组(0.95±0.26)、二联苯碘干预组(0.99±0.28)、SB203580干预组(0.93±0.31)、LY294002干预组(1.07±0.19)均低于PDGF组,差异均有统计学意义(t=4.209、-2.234、-4.424、-4.252、-4.510、-3.909,P均＜0.05).PI3K蛋白表达水平,PDGF组(2.27±0.46)高于空白对照组(1.00±0.00),熊果酸干预组(0.59±0.28)低于PDGF组和空白对照组,熊果酸对照组(0.14±0.07)低于空白对照组,二联苯碘干预组(0.53±0.25)、LY294002干预组(0.35±0.14)均低于PDGF组,差异均有统计学意义(t=6.205、-8.208、-2.003、-4.202、-8.502、-9.831,P均＜0.05).p-Akt蛋白表达水平,PDGF组(2.54±0.49)高于空白对照组(1.00±0.00),熊果酸干预组(0.74±0.20)、二联苯碘干预组(0.94±0.37)、LY294002干预组(1.17±0.41)均低于PDGF组,熊果酸对照组(0.59±0.15)低于空白对照组,差异均有统计学意义(t=5.927、-6.928、6.158、-5.273、-1.578,P均＜0.05).磷酸化p38MAPK蛋白表达水平,PDGF组(1.98±0.35)高于空白对照组(1.00±0.00),熊果酸干预组(0.68±0.28)、二联苯碘干预组(0.63±0.27)、SB203580干预组(0.67±0.29)均低于PDGF组,熊果酸对照组(0.28±0.13)低于空白对照组,差异均有统计学意义(t=4.897、-6.479、-6.727、-6.529、-3.561,P均＜0.05).活性氧水平,PDGF组(105.57±7.51)高于空白对照组(69.60±8.63),熊果酸干预组(64.56±9.11)、二联苯碘干预组(65.75±6.62)均低于PDGF组,熊果酸对照组(29.84±3.19)低于空白对照组,差异均有统计学意义(t=6.368、-7.288、-7.071、-7.255,P均＜0.05).结论 熊果酸或可抑制PDGF诱导的大鼠HSC-T6细胞内NOX亚基p47phox蛋白膜移位及活性氧的产生,进而阻断PI3K-蛋白激酶B、p38MAPK信号通路的磷酸化及Ⅰ型胶原mRNA的表达.
目的 觀察熊果痠對血小闆衍生生長因子(PDGF)誘導的大鼠活化型肝星狀細胞株(HSC-T6)還原型煙酰胺腺嘌呤二覈苷痠燐痠氧化酶(NOX)活化及下遊信號通路激活的影響.方法 取大鼠HSC-T6,分為空白對照組(不予處理)、熊果痠對照組(50 μmol/L)、PDGF組(10 μg/L)、熊果痠榦預組(50 μ mol/L熊果痠+10μg/L PDGF)、二聯苯碘榦預組(20μmol/L二聯苯碘+10 μg/L PDGF)、SB203580榦預組(10 μmol/L SB203580+ 10 μg/LPDGF)、LY294002榦預組(10 μmol/L LY294002+10 μg/L PDGF)、活性氧暘性對照組(5μg/mL活性氧試劑rosup).採用熒光定量-PCR法檢測除活性氧暘性對照組外的其餘各組細胞中Ⅰ型膠原mRNA的錶達水平.採用Western印跡法檢測膜蛋白NOX亞基p47phox(除外活性氧暘性對照組)、燐脂酰肌醇3激酶(PI3K,除外活性氧暘性對照組和SB203580榦預組)、燐痠化蛋白激酶B(p-Akt,除外活性氧暘性對照組和SB203580榦預組)、燐痠化p38絲裂原活化蛋白激酶(除外活性氧暘性對照組和LY294002榦預組)的錶達.採用活性氧檢測試劑盒和熒光酶標儀檢測除SB203580榦預組和LY294002榦預組外的其餘各組細胞內熒光彊度.組間比較行單因素方差分析和LSD檢驗.結果 Ⅰ型膠原mRNA錶達水平,PDGF組(3.74±0.32)高于空白對照組(1.00土0.00),熊果痠對照組(0.21±0.02)低于空白對照組,熊果痠榦預組(1.02±0.12)、二聯苯碘榦預組(1.09±0.21)、SB203580榦預組(1.18±0.27)、LY294002榦預組(1.15±0.26)均低于PDGF組,差異均有統計學意義(t=15.667、-4.501、-15.553、-15.154、-14.642、-14.813,P均＜0.05).p47phox蛋白錶達水平,PDGF組(1.98±0.53)高于空白對照組(1.00±0.00),熊果痠對照組(0.48±0.10)低于空白對照組,熊果痠榦預組(0.95±0.26)、二聯苯碘榦預組(0.99±0.28)、SB203580榦預組(0.93±0.31)、LY294002榦預組(1.07±0.19)均低于PDGF組,差異均有統計學意義(t=4.209、-2.234、-4.424、-4.252、-4.510、-3.909,P均＜0.05).PI3K蛋白錶達水平,PDGF組(2.27±0.46)高于空白對照組(1.00±0.00),熊果痠榦預組(0.59±0.28)低于PDGF組和空白對照組,熊果痠對照組(0.14±0.07)低于空白對照組,二聯苯碘榦預組(0.53±0.25)、LY294002榦預組(0.35±0.14)均低于PDGF組,差異均有統計學意義(t=6.205、-8.208、-2.003、-4.202、-8.502、-9.831,P均＜0.05).p-Akt蛋白錶達水平,PDGF組(2.54±0.49)高于空白對照組(1.00±0.00),熊果痠榦預組(0.74±0.20)、二聯苯碘榦預組(0.94±0.37)、LY294002榦預組(1.17±0.41)均低于PDGF組,熊果痠對照組(0.59±0.15)低于空白對照組,差異均有統計學意義(t=5.927、-6.928、6.158、-5.273、-1.578,P均＜0.05).燐痠化p38MAPK蛋白錶達水平,PDGF組(1.98±0.35)高于空白對照組(1.00±0.00),熊果痠榦預組(0.68±0.28)、二聯苯碘榦預組(0.63±0.27)、SB203580榦預組(0.67±0.29)均低于PDGF組,熊果痠對照組(0.28±0.13)低于空白對照組,差異均有統計學意義(t=4.897、-6.479、-6.727、-6.529、-3.561,P均＜0.05).活性氧水平,PDGF組(105.57±7.51)高于空白對照組(69.60±8.63),熊果痠榦預組(64.56±9.11)、二聯苯碘榦預組(65.75±6.62)均低于PDGF組,熊果痠對照組(29.84±3.19)低于空白對照組,差異均有統計學意義(t=6.368、-7.288、-7.071、-7.255,P均＜0.05).結論 熊果痠或可抑製PDGF誘導的大鼠HSC-T6細胞內NOX亞基p47phox蛋白膜移位及活性氧的產生,進而阻斷PI3K-蛋白激酶B、p38MAPK信號通路的燐痠化及Ⅰ型膠原mRNA的錶達.
목적 관찰웅과산대혈소판연생생장인자(PDGF)유도적대서활화형간성상세포주(HSC-T6)환원형연선알선표령이핵감산린산양화매(NOX)활화급하유신호통로격활적영향.방법 취대서HSC-T6,분위공백대조조(불여처리)、웅과산대조조(50 μmol/L)、PDGF조(10 μg/L)、웅과산간예조(50 μ mol/L웅과산+10μg/L PDGF)、이련분전간예조(20μmol/L이련분전+10 μg/L PDGF)、SB203580간예조(10 μmol/L SB203580+ 10 μg/LPDGF)、LY294002간예조(10 μmol/L LY294002+10 μg/L PDGF)、활성양양성대조조(5μg/mL활성양시제rosup).채용형광정량-PCR법검측제활성양양성대조조외적기여각조세포중Ⅰ형효원mRNA적표체수평.채용Western인적법검측막단백NOX아기p47phox(제외활성양양성대조조)、린지선기순3격매(PI3K,제외활성양양성대조조화SB203580간예조)、린산화단백격매B(p-Akt,제외활성양양성대조조화SB203580간예조)、린산화p38사렬원활화단백격매(제외활성양양성대조조화LY294002간예조)적표체.채용활성양검측시제합화형광매표의검측제SB203580간예조화LY294002간예조외적기여각조세포내형광강도.조간비교행단인소방차분석화LSD검험.결과 Ⅰ형효원mRNA표체수평,PDGF조(3.74±0.32)고우공백대조조(1.00토0.00),웅과산대조조(0.21±0.02)저우공백대조조,웅과산간예조(1.02±0.12)、이련분전간예조(1.09±0.21)、SB203580간예조(1.18±0.27)、LY294002간예조(1.15±0.26)균저우PDGF조,차이균유통계학의의(t=15.667、-4.501、-15.553、-15.154、-14.642、-14.813,P균＜0.05).p47phox단백표체수평,PDGF조(1.98±0.53)고우공백대조조(1.00±0.00),웅과산대조조(0.48±0.10)저우공백대조조,웅과산간예조(0.95±0.26)、이련분전간예조(0.99±0.28)、SB203580간예조(0.93±0.31)、LY294002간예조(1.07±0.19)균저우PDGF조,차이균유통계학의의(t=4.209、-2.234、-4.424、-4.252、-4.510、-3.909,P균＜0.05).PI3K단백표체수평,PDGF조(2.27±0.46)고우공백대조조(1.00±0.00),웅과산간예조(0.59±0.28)저우PDGF조화공백대조조,웅과산대조조(0.14±0.07)저우공백대조조,이련분전간예조(0.53±0.25)、LY294002간예조(0.35±0.14)균저우PDGF조,차이균유통계학의의(t=6.205、-8.208、-2.003、-4.202、-8.502、-9.831,P균＜0.05).p-Akt단백표체수평,PDGF조(2.54±0.49)고우공백대조조(1.00±0.00),웅과산간예조(0.74±0.20)、이련분전간예조(0.94±0.37)、LY294002간예조(1.17±0.41)균저우PDGF조,웅과산대조조(0.59±0.15)저우공백대조조,차이균유통계학의의(t=5.927、-6.928、6.158、-5.273、-1.578,P균＜0.05).린산화p38MAPK단백표체수평,PDGF조(1.98±0.35)고우공백대조조(1.00±0.00),웅과산간예조(0.68±0.28)、이련분전간예조(0.63±0.27)、SB203580간예조(0.67±0.29)균저우PDGF조,웅과산대조조(0.28±0.13)저우공백대조조,차이균유통계학의의(t=4.897、-6.479、-6.727、-6.529、-3.561,P균＜0.05).활성양수평,PDGF조(105.57±7.51)고우공백대조조(69.60±8.63),웅과산간예조(64.56±9.11)、이련분전간예조(65.75±6.62)균저우PDGF조,웅과산대조조(29.84±3.19)저우공백대조조,차이균유통계학의의(t=6.368、-7.288、-7.071、-7.255,P균＜0.05).결론 웅과산혹가억제PDGF유도적대서HSC-T6세포내NOX아기p47phox단백막이위급활성양적산생,진이조단PI3K-단백격매B、p38MAPK신호통로적린산화급Ⅰ형효원mRNA적표체.
Objective To observe the effects of ursolic acid (UA) on the activation of nicotinamide adenine dinucleotide phosphate oxidase (NOX) and the downstream signaling pathways in platelet derived growth factor (PDGF) activated rat hepatic stellate cell (HSC-T6).Methods Rat HSC-T6 cells were divided into blank control group (no treatment),UA control group (50 μmol/L UA),PDGF group (10 μg/L PDGF),UA intervention group (50 μmol/L UA + 10 μg/L PDGF),diphenyleneiodonium intervention(DPI) group (20 μmol/L DPI+ 10 μg/L PDGF),SB203580 (p38 mitogen-activated protein kirase(p38MAPK) inhibitor) intervention group (10 μmol/L SB203580 + 10 μg/LPDGF),LY294002 (phosphatidylinositop 3 kinase(PI3K) inhibitor) intervention group (10 μmol/L LY294002 + 10 μg/L PDGF) and rosup positive control group (5 μg/mL rosup).Except rosup positive control group,the expression of type Ⅰ collagen at mRNA level of each group was detected by fluorescence quantitavepolymerase chain reaction (RT-PCR).The expression of membrane protein p47phox (except rosup positive control group),PI3K(except rosup positive control group and SB203580 intervention group),p-protein kinase B (p-AKT,except rosup positive control group and SB203580 intervention group) and phosphorylated p38 mitogen-activated protein kinase (p-p38MAPK,except rosup positive control group and LY294002 intervention group) were tested by Western blot.Except SB203580 intervention group and LY294002 intervention group,the fluorescence intensity in the cells of each group was analyzed with active oxygen detection kit and fluorescence microplate reader.Single factor analysis of variance and LSD test were performed for comparison between groups.Results Type Ⅰ collagen at the mRNA level of PDGF group (3.74±0.32) was higher than that of blank control group (1.00±0.00) ; Type Ⅰ collagen at the mRNA level of UA group (0.21 ±0.02) was lower than that of blank control group,UA intervention group (1.02 ± 0.12),DPI intervention group (1.09±0.21),SB203580 intervention group (1.18± 0.27),and LY294002 intervention group (1.15 ± 0.26) were all lower than PDGF group,and the differences were statistically significant (t =15.667,-4.501,-15.553,-15.154,-14.642 and -14.813,all P＜0.05).p47phox at the protein expression level of PDGF group (1.98±0.53) was higher than that of blank control group (1.00±0.00) ; that of UA group (0.48±0.10) was lower than blank control group; those of UA intervention group (0.95 ± 0.26),DPI intervention group (0.99 ± 0.28),SB203580 intervention group (0.93±0.31),and LY294002 intervention group (1.07±0.19) were all lower than PDGF group (t=4.209,-2.234,4.424,-4.252,-4.510 and-3.909,all P＜0.05).The protein expression level of PI3K of PDGF group (2.27±0.46) was higher than that of blank control group (1.00±0.00); that of UA intervention group (0.14 ± 0.07) was lower than PDGF group and blank control group; that of UA group (0.14±0.07) was lower than blank control group; those of DPI intervention group (0.53±0.25) and LY294002 intervention group (0.35±0.14) were all lower than PDGFgroup (t 6.205,8.208,-2.003,4.202,-8.502 and-9.831,all P＜0.05).The protein expression level of p-Akt of PDGF group (2.54±0.49) was higher than that of blank control group (1.00± 0.00); those of UA intervention group (0.74± 0.20),DPI intervention group (0.94 ± 0.37) and LY294002 intervention group (1.17±0.41) were all lower than PDGF group; that of UA group (0.59± 0.15) was lower than blank control group (t=5.927,-6.928,-6.158,-5.273 and-1.578,all P＜ 0.05).The protein expression level of p-p38MAPK of PDGF group (1.98±0.35) was higher than that of blank control group (1.00±0.00); those of UA intervention group (0.68±0.28),DPI intervention group (0.63±0.27) and SB203580 intervention group (0.67 ± 0.29) was all lower than PDGF group; that of UAgroup (0.28±0.13) was lower than blank control group (t=4.897,-6.479,-6.727,-6.529 and-3.561,all P＜0.05).The level of active oxygen of PDGF group (105.57±7.51) was higher than that of blank control group (69.60±8.63) ; those of UA intervention group (64.56±9.11),DPI intervention group (65.75 ± 6.62) was lower than PDGF group,UA group (29.84 ±3.19) was lower than blank control group (t=6.368,-7.288,-7.071 and-7.255,all P＜0.05).Conclusion UA could inhibit membrane displacement of NOX subunit p47phox and reduce active oxygen production in PDGF induced rat HSC-T6 cells,and then block phosphorylation of PI3K Akt,p 38MAPK signal pathways and inhibited the expression of type Ⅰ collagen at mRNA level.
