The Experimental Study Of The Effects Of Astragalus Mongholicus Bge On Hela Cells' Proliferation And Related Regulators Expression

Objective:Malignant tumour has been one of the most important diseases endangering people' lives and health,about one million people die of malignant tumour every year.We have got certain achievement on the research of pathogenesis and therapy,and more and more focus was emphasized on the combined therapy,especially the research of traditional Chinese medicines on the treatment and co-treatment of tumour. Previous research of traditional Chinese medicine mostly aimed at inhibiting the growth of tumour,and there are few reports on promoting their growth and proliferation.It was found that Astragalus Mongholicus Bge(AMB) can promote uterine cervix cancer Hela cells' growth and proliferation in preliminary experiment,which has been used in clinic to invigorate vital energy and to adjuvently treat tumour as the traditional Chinese medicines on strengthening healthy and eliminating pathogens. So it indicated that diverge still exsisted on the possibility of AMB and extractive used on the treatment of patients with uterine cervix cancer and other patients susceptibility of this tumor.Our study planned to discuss the effects of AMB on Hela cells' proliferation and the expression of the related regulators, to provide experimental basement and theoretic evidences to guide the proper application for clinical uses of this kind of traditional Chinese medicine.Methods:The suitable inoculated concentration and cultured time of Hela cells were detected by MTT.The Hela cells treated with AMB for 48h,and the corresponding Hela cells treated without AMB as control were used.The A₄₉₂ value of Hela cells treated by different concentrations of AMB were measured;The cell cycle and the state of proliferation and apoptosis were directed by FCM to calculate the apoptotic index and proliferation index;The cultured cells were stained by hematoxylin eosin for routinely pathological observation;the expression of PCNA,Bax,Bcl-2 in Hela cells treated by different concentrations of AMB were analyzed with immunocytochemical staining,200 cells were counted in rectangle to calculate the percentage of positive cells,and were analyzed by image analysis system synchronously,the area of positive staining was showed by positive enzyme dot(PED),the indensity of positive staining was showed by grey scale(GS) (the value range of GS was 0～255,larger value of GS reflects the weaker indensity of positive staining,and the smaller value of GS reflects the stronger indensity of positive staining).Results:1 The effect of AMB on the proliferation of Hela cells The inoculated concentration determined by MTT was 2×10⁵/mL,the culture time was 48 hours;compared with the control group(0.973±0.073),the A₄₉₂ of AMB of 2000 mg/L,1000 mg/L,500 mg/L,250 mg/L and 125 mg/L elevated greatly and gradually(1.427±0.158,P＜0.01;1.522±0.194, P＜0.01;1.275±0.125,P＜0.01;1.219±0.116,P＜0.01, respectively),the A₄₉₂ of AMB of 62.5 mg/L and 31.25 mg/L had no difference(1.052±0.111,1020±0.089,all P＞0.05, respectively).2 The effects of AMB on the cell cycle,apoptosis and proliferation of Hela cells2.1 The effects on the cell cycleCompared with control group(68.28%±2.01%),the proportion of G0/G1 stage of AMB 2000 mg/L,1000 mg/L,500 mg/L,250 mg/L,125 mg/L,62.5 mg/L and 31.25 mg/L were lower obviously(59.64±2.77,P＜0.05;58.96±2.67,P＜0.05; 60.49±2.89,P＜0.05;63.84±3.26,P＜0.05;61.68±3.15, P＜0.05;57.83±2.34,P＜0.01;57.39±2.46,P＜0.01, respectively).Compared with the control group(13.49%±1.96%),the proportion of S stage of AMB 2000 mg/L,1000 mg/L,500 mg/L,250 mg/L,125 mg/L,62.5 mg/L and 31.25 mg/L were higher obviously(19.35%±1.53%,P＜0.01;16.06%±0.82%, P＜0.05;16.33%±0.85%,P＜0.05;15.97%±0.74%,P＜0.05; 17.79%±1.15%,P＜0.05;17.89%±1.21%,P＜0.05;17.65% ±0.97%,P＜0.05,respectively).2.2 The effects on apoptosis and proliferationCompared with control group(5.41%±0.61%),the AI of AMB 2000 mg/L,1000 mg/L,500 mg/L,250 mg/L,125 mg/L,62.5 mg/L and 31.25 mg/L were lower obviously(3.32%±0.05%,P＜0.01;4.22%±0.12%,P＜0.05;4.22%±0.12%, P＜0.05;2.88%±0.23%,P＜0.01;2.81%±0.20%,P＜0.01; 6.29%±0.27%,P＜0.05;4.98±0.20,P＜0.05,respectively). AI in cells treated with AMB of 2000 mg/L and 31.25 mg/L had significant difference with other groups;there is no significant difference among 1000 mg/L,500 mg/Land 62.5 mg/L of AMB; there was no significant difference between 250 mg/L and 125 mg/L.Compared with control group(29.85%±2.65%),the PI of AMB 2000 mg/L,1000 mg/L,500 mg/L,250 mg/L were higher obviously(37.16%±4.13%,42.75%±4.52%,36.68%±3.34%,36.56%±3.05%,all P＜0.05,respectively),the PI of AMB 125 mg/L,62.5 mg/L及31.25 mg/L had no difference (35.58%±2.96%,35.25%±3.14%,35.28%±3.22%,all P＞0.05,respectively).The mean comparison of interclassevery was that there was significant difference between 1000 mg/L of AMB with other groups,there was no significant difference among 2000 mg/L,500 mg/L,250 mg/L,125 mg/L,62.5 mg/L and 31.25 mg/L of AMB.3 The effects of AMB on the expression of related regulators3.1 The effect of AMB on the expression of Bax3.1.1 The positive percent of Bax:compared with control group(36.17%±3.05%),the positive percent of AMB 2000 mg/L,1000 mg/L,500 mg/L,250 mg/L,125 mg/L,62.5 mg/L and 31.25 mg/L were lower obviously of Bax in Hela cells(14.24%±1.76%,19.70%±1.89%,21.21%±2.17%,23.67%±2.29%, 25.06%±2.31%,28.45%±2.42%,30.52%±2.33%,all P＜0.05, respectively).The correlation analysis was significant negative correlation between positive percent and concentration(r=-0.815,P=0.000).3.1.2 PED of Bax:compared with control group(481.49±58.34),the PED of AMB 2000 mg/L,1000 mg/L,500 mg/L,250 mg/L,125mg/L,62.5mg/L and 31.25mg/L were lower obviously of bax in Hela cells(246.57±36.78,P＜0.01;257.09±40.00,P＜0.01;289.34±39.63,P＜0.01;376.69±28.71, P＜0.01;334.46±33.38,P＜0.01;386.31±54.91,P＜0.05; 392.86±42.01,P＜0.05,respectively).The correlation analysis was significant negative correlation between PED and concentration(r=-0.714,P=0.000),significant positive correlation between PED and positive percent(r =0.946,P=0.000).3.1.3 GS of Bax:compared with control group(121.13±7.97),the GS of AMB 2000 mg/L,1000 mg/L,500 mg/L,250 mg/L higher obviously of bax in Hela cells(159.03±20.82, 146.45±12.17,143.37±11.31,152.81±14.40,P＜0.05 respectively),the GS of AMB 125 mg/L,62.5 mg/L and 31.25 mg/L were no difference(127.01±5.49,128.62±9.66,123.90±9.50,P＞0.05,respectively).The correlation analysis was significant positive correlation between GS and concentration(r =0.636,P=0.001).3.2 The effect of AMB on the expression of Bcl-23.2.1 The positive percent of Bcl-2:compared with control group(79.85%±2.47%),the positive percent of AMB 2000 mg/L,1000 mg/L,500 mg/L,250 mg/L were higher obviously of Bcl-2 in Hela cells(96.02%±3.88%,89.73%±3.25%,92.34%±3.54%,94.65%±3.69%,P＜0.05,respectively),the positive percent of AMB 125 mg/L,62.5 mg/L and 31.25 mg/L were no difference(84.22%±3.18%,82.56%±2.98%,80.32%±2.61%, all P＞0.05,respectively).The correlation analysis was significant positive correlation between positive percent and concentration(r=0.658,P=0.000).3.2.2 PED of Bcl-2:compared with control group(718.40±94.94),the PED of AMB 2000 mg/L,1000 mg/L,500 mg/L,250 mg/L were higher obviously of Bcl-2 in Hela cells (1292.60±147.54,P＜0.01;1124.74±139.64,P＜0.01;1168.40±96.69,P＜0.01;866.54±72.40,P＜0.05,respectively),the PED of AMB 125mg/L,62.5mg/L and 31.25mg/L were no difference (852.63±81.65,859.31±74.02,753.34±72.95,all P＞0.05, respectively).The correlation analysis was significant positive correlation between PED and concentration(r=0.815,P=0.000), significant positive correlation between PED and positive percent(r=0.688,P=0.000)3.2.3 GS of Bcl-2:compared with control group(133.56±2.89),the GS of AMB 2000 mg/L,1000 mg/L,500 mg/L,250 mg/L,125mg/L,62.5mg/L and 31.25mg/L were lower obviously of Bcl-2 in Hela cells(90.06±7.10,98.78±5.55, 103.47±5.19,104.37±5.38,100.00±8.71,97.01±8.96, 108.84±5.17,133.56±2.89,all P＜0.01,respectively).The correlation analysis was significant negative correlation between GS and concentration(r=-0.535,P=0.007)3.3 The effect of AMB on the expression of PCNA3.3.1 The positive percent of PCNA:compared with control group(42.71%±2.01%),the positive percent of AMB 2000 mg/L,1000 mg/L,500 mg/L,250 mg/L,125 mg/L and 62.5 mg/L were higher obviously of PCNA in Hela cells(69.28±4.01,64.56±3.846,62.27±3.69,54.02±3.51,57.34±3.28, 48.40±3.26,all P＜0.05,respectively),the positive percent of AMB 31.25 mg/L was no difference(46.96±2.04,P＞0.05). The correlation analysis was significant positive correlation between positive percent and concentration(r=0.810,P=0.000).3.3.2 PED of PCNA:compared with control group(249.57±40.23),the PED of AMB 2000 mg/L,1000 mg/L,500 mg/L,250 mg/L,125 mg/L,62.5 mg/L and 31.25 mg/L were higher obviously of PCNA in Hela cells(523.91±64.66, P＜0.01;497.94±42.76,P＜0.01;499.40±40.29,P＜0.01; 449.03±46.85,P＜0.01;400.06±60.12,P＜0.01;339.89±45.39,P＜0.05;350.03±40.92,P＜0.05,respectively).The correlation analysis was significant positive correlation between PED and concentration(r=0.683,P=0.000),significant positive correlation between PED and positive percent(r =0.947,P=0.000).3.3.3 GS of PCNA:compared with control group(155.50±18.02),the GS of AMB 2000 mg/L,1000 mg/L,500 mg/L,250 mg/L,125 mg/L,62.5 mg/L and 31.25 mg/L were no difference (150.67±13.69,150.49±13.46,152.37±10.24,139.08±16.18, 160.49±14.68,155.01±15.12,156.77±22.65,all P＞0.05 respectively).The correlation analysis was that the correlation is no difference between GS and concentration(r=-0.108,P=0.616)Conclusions:1.AMB inhibited the apoptosis and promote proliferation of Hela cells within certain extent of concentration.2.AMB blocked the cell cycle in S stage of Hela cells within certain extent of concentration.3.AMB enhanced the expression of Bcl-2 and PCNA, depressed the expression of Bax in Hela cells within certain extent of concentration.4.AMB should be used carefully in clinical treatment of tumor and other diseases.