| Objective: Cervical cancer is the main death cause of women cancer, its incidence rate is second followed by the breast cancer. Presently, the main therapies of cervical cancer include surgery and chemoradiation therapy, but the traditional Chinese medicine can improve the postoperative damnification and subsidiary reaction of chemoradiation therapy. As a kind of preparation of activating blood circulation to dissipate blood stasis, Salvia Miltiorrhiza Bunge (SMB) has been used to many clinical diseases such as cerebrovascular disease, neurogenic disease, hepatitis and liver fibrosis, more and more study was emphasized on the treatment of tumour.But it was found that SMB can promote cervical cancer Hela cells'growth and proliferation in our preliminary experiment. So it indicated that diverge still exsisted on the possibility of SMB and its extractive which used on the treatment of patients with cervical cancer and other patients susceptibility of this tumor. Our study planned to discuss the effects of SMB on Hela cells'proliferation and the expression of 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 SMB for 48h, and the corresponding Hela cells treated without SMB as control were used. The A492 value of Hela cells treated by different concentrations of SMB 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 SMB 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 density 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 SMB on the proliferation of Hela cellsThe inoculated concentration determined by MTT was 2×105/mL, the culture time was 48 hours; compared with the control group(0.953±0.093),the A492 of SMB of 200mg/mL, 100mg/mL, 50mg/mL, 25mg/mL and 12.5mg/mL SMB elevated greatly and gradually (1.957±0.102, 1.853±0.087, 1.743±0.107, 1.616±0.102, 1.776±0.185, all P<0.01, respectively), the A492 of SMB of 6.25mg/mL and 3.125mg/mL had no difference (1.118±0.230, 1.014±0.182, all P>0.05, respectively). The correlation analysis was positive correlation between A492 and concentration(r=0.707, P=0.05).2 The effects of SMB on the cell cycle, apoptosis and proliferation of Hela cells2.1 The effects on the cell cycleCompared with the control group(79.47%±0.47%), the proportion of G0/G1 stage of SMB 200mg/mL, 100mg/mL, 50mg/mL, 25mg/mL and 12.5mg/mL were lower obviously (58.56%±2.43%, 61.88%±2.38%, 65.67%±1.25%, 64.89%±2.45%, 64.82%±3.24%, all P<0.05, respectively), the proportion of G0/G1 stage of SMB 6.25mg/mL and 3.125mg/mL had no difference(72.94%±8.23%, 76.31%±2.67%, all P>0.05).Compared with the control group(13.22%±1.89%), the proportion of S stage of SMB 200mg/mL, 100mg/mL, 50mg/mL, 25mg/mL and 12.5mg/mL were higher obviously (23.80%±3.90%, P<0.01; 24.60%±3.60%, P<0.01; 20.98%±1.35%, P<0.01; 20.12%±1.12%, P<0.01; 19.04%±0.62%, P<0.05, respectively), the proportion of S stage of SMB 6.25mg/mL and 3.125mg/mL had no difference(17.92%±1.09%, 16.59%±0.78%, all P>0.05). 2.2 The effects on apoptosis and proliferationCompared with the control group(5.85%±1.44%), the AI of SMB 200mg/mL, 100mg/mL, 50mg/mL and 25mg/mL were lower obviously (1.20%±0.06%, P<0.01; 2.24%±0.72%,P<0.01; 2.82%±0.42%, P<0.01; 3.54%±0.60%, P<0.05, respectively), the AI of SMB 12.5mg/mL, 6.25mg/mL and 3.125mg/mL had no difference(4.45%±1.74%, P <0.01; 5.32%±0.87%, P<0.05; 5.74%±0.78%, P<0.05, respectively). The correlation analysis was negative correlation between AI and concentration(r =-0.892, P=0.003).Compared with the control group(13.13%±2.75%), the PI of SMB 200mg/mL, 100mg/mL and 50mg/mL were higher obviously (31.86%±1.45%, P<0.01; 27.54%±1.08%, P<0.01; 18.73%±2.51%, P<0.05, respectively), the PI of SMB 25mg/mL, 6.25mg/mL and 3.125mg/mL had no difference (16.13%±2.02%, 16.36%±2.96%, 15.77%±2.62%, 13.69%±3.12%, all P>0.05). The correlation analysis was positive correlation between PI and concentration(r =0.969, P=0.000).3 The effects of SMB on the expression of related regulators3.1 The effect of SMB on the expression of Bax3.1.1 The positive percent of Bax: compared with control group(35.16%±2.75%), the positive percent of SMB 200mg/mL, 100mg/mL, 50mg/mL, 25mg/mL and 12.5mg/mL were lower obviously of Bax in Hela cells (15.50%±1.50%, 19.66%±1.75%, 24.66%±2.25%, 21.50%±2.00%, 28.16%±2.25%, respectively, all P<0.05), the positive percent of SMB 6.25mg/mL and 3.125mg/mL had no difference (31.16%±2.25%, 33.16%±2.50%, all P>0.05). The correlation analysis was negative correlation between positive percent and concentration(r =-0.851, P=0.007).3.1.2 GS of Bax: compared with control group (85.07±15.31), the GS of SMB 200mg/mL, 100mg/mL were higher obviously of bax in Hela cells (140.95±8.51, 136.45±32.33, P<0.05 respectively), the GS of SMB 50mg/mL, 25mg/mL, 12.5mg/mL, 6.25mg/mL and 3.125mg/mL were no difference (122.85±15.95, 119.44±30.13, 108.50±16.45, 98.81±0.50, 95.95±5.00, P>0.05, respectively). The correlation analysis was positive correlation between GS and concentration(r =0.850, P=0.007).3.2 The effect of SMB on the expression of Bcl-23.2.1 The positive percent of Bcl-2: compared with the control group(77.96%±2.55%), the positive percent of SMB 200mg/mL, 100mg/mL, 50mg/mL and 25mg/mL were higher obviously of Bcl-2 in Hela cells (95.83%±3.75%, 94.00%±3.50%, 88.50%±3.00%, 91.16%±3.25%, all P<0.01, respectively), the positive percent of SMB 12.5mg/mL, 6.25mg/mL and 3.125mg/mL were no difference(85.16%±2.75%, 80.50%±2.78%, 82.36%±2.75%, all P>0.05). The correlation analysis was positive correlation between positive percent and concentration(r =0.821, P=0.012).3.2.2 GS of Bcl-2: compared with control group (144.21±14.26), the GS of SMB 200mg/mL, 100mg/mL, 50mg/mL and 25mg/mL were lower obviously of Bcl-2 in Hela cells (105.18±1.20, 109.08±3.78, 113.15±8.54, 112.81±12.04, all P<0.01, respectively), the GS of SMB 12.5mg/mL, 6.25mg/mL and 3.125mg/mL were no difference (129.84±6.03, 127.59±2.63, 144.59±9.45, all P>0.05).The correlation analysis was negative correlation between GS and concentration(r =-0.747, P=0.033).3.3 The effect of SMB on the expression of PCNA3.3.1 The positive percent of PCNA: compared with the control group(41.00%±2.00%), the positive percent of SMB 200mg/mL, 100mg/mL, 50mg/mL and 25mg/mL were higher obviously of PCNA in Hela cells (65.16%±3.75%, 60.33%±3.75%, 57.16%±3.75%, 53.66%±3.25%, all P<0.01, respectively), the positive percent of SMB 12.5mg/mL, 6.25mg/mL and 3.125mg/mL were no difference(45.33%±2.75%, 43.00%±2.50%, 47.33%±3.25%, all P>0.05). The correlation analysis was positive correlation between positive percent and concentration(r =0.881, P=0.004).3.3.2 GS of PCNA: compared with the control group (153.999±5.18), the GS of SMB 200mg/mL, 100mg/mL, 50mg/mL and 25mg/mL were higher obviously of PCNA in Hela cells (122.05±9.70, 133.76±6.34, 134.22±7.76, 138.81±13.01, all P<0.05, respectively). the GS of 12.5mg/mL, 6.25mg/mL and 3.125mg/mL were no difference(146.50±4.48, 157.15±5.07, 157.31±4.52, all P>0.05). The correlation analysis was negative correlation between positive percent and concentration(r =-0.877, P=0.004).Conclusions:1 SMB inhibited the apoptosis and promote proliferation of Hela cells within certain extent of concentration.2 SMB blocked the cell cycle in S stage of Hela cells within certain extent of concentration.3 SMB enhanced the expression of Bcl-2 and PCNA, depressed the expression of Bax in Hela cells within certain extent of concentration.4 SMB should be used carefully in clinical treatment of tumor and other diseases.
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