图1 不同浓度黄芩素对MC3T3-E1 细胞增殖的影响
纸质出版日期:2024-07-20,
收稿日期:2024-04-25,
录用日期:2024-06-13
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研究不同浓度黄芩素对MCT3-E1增殖及生物学行为的影响,以及其对口腔常见菌的抑菌作用,并探讨其相关机制。
将MC3T3-E1细胞分别培养在0、6、12、18、24 μmol/L的黄芩素浓度下,通过CCK-8实验检测黄芩素处理后MC3T3-E1的增殖活性;对成骨诱导培养后的MC3T3-E1进行ALP活性检测;RT-PCR法检测RunX2、BMP2、Osterix基因的表达差异。K-B纸片法检测黄芩素处理24 h对大肠杆菌,金黄色葡萄球菌,血链球菌的抑菌效果。
黄芩素可在一定程度降低细胞增殖活力,但不影响其继续增殖。18 μmol/L的黄芩素可以提高MC3T3-E1的ALP活性,并有效上调BMP2与Osterix的表达,下调RunX2的表达,能有效抑制金黄色葡萄球菌与血链球菌的增殖(P<0.05)。
适宜浓度的黄芩素(18 μmol/L)对MC3T3-E1细胞成骨分化有促进作用,并且可有效抑制口腔常见菌(金黄色葡萄球菌与血链球菌)的增殖。
To investigate the impact of varying concentrations of baicalein on the proliferation and biological responses of MC3T3-E1 cells, as well as the antibacterial efficacy of baicalein against prevalent oral bacteria, and to elucidate the underlying mechanisms.
MC3T3-E1 cells were exposed to different concentrations of baicalein (0, 6, 12, 18, and 24 μmol/L) and cell viability was determined by using the CCK-8 assay. Alkaline phosphatase (ALP) activity of MC3T3-E1 cells following osteogenic induction was assessed. RT-PCR was used to examine the expression of RunX2, BMP2, and Osterix. After 24 hours of treatment, the antibacterial potential of baicalein against Escherichia coli, Staphylococcus Aureus and Streptococcus Sanguis was evaluated by using the K-B paper disk method.
Baicalein exhibited a modest reduction in proliferation of MC3T3-E1 cells but without affecting their sustained proliferation. Baicalein at a concentration of 18 μmol/L enhanced ALP activity of MC3T3-E1 cells, upregulated BMP2 and Osterix expression, downregulated RunX2 expression, significantly inhibited the proliferation of Staphylococcus Aureus and Streptococcus Sanguis (P < 0.05).
Baicalein at an optimal concentration (18 μmol/L) demonstrated a promotional effect on the osteogenic differentiation of MC3T3-E1 cells and effectively suppressed the proliferation of common oral bacteria, including Staphylococcus Aureus and Streptococcus Sanguis.
口腔常见疾病牙周病、种植体周围炎、微种植体周围炎均是以微生物为始动因子,局部组织发生慢性炎症性反应,牙体支持组织逐渐破坏丧失,牙槽骨逐渐吸收的疾病。对于此类牙周支持组织疾病治疗的长期目标主要是清除口腔内菌斑,保持口腔卫生,同时阻止局部炎症继续发展,阻断骨吸收,尽可能恢复原有的骨量。药物辅助治疗牙周支持组织炎症是常见的辅助治疗手段,近年来多有文献提及以中药治疗此类疾病。黄芩素是从黄芩根部主要提取出的一种天然黄酮类化合物。其性状为黄色的针状结晶,化学名称为 5,6,7-三羟基黄酮,分子量为 270.2,在中药领域已有上千年的历史,已被证实在口腔颌面部肿瘤、黏膜疾病、硬组织疾病、炎症性疾病中均发挥重要作用[
小鼠前成骨细胞MC3T3-E1细胞(广州威佳生物科技有限公司,中国)。黄芩素(乐美天医药/德思特生物有限公司,中国);CCK-8 试剂盒(广州威佳生物科技有限公司,中国);OPTI-MEM(Invitrogen,美国);FBS(ExCell Bio,中国);α-MEM(GIBCO,中国);胰蛋白酶(ECOTOP,英国);STAT3 抗体(abcam,中国);Thapsigargin(MCE,美国);MK-28(MCE,美国);DL2000(TaKaRa,日本);1 kbp DNA Ladder Marker(TaKaRa,日本);PrimeSTAR HS DNA-Polymerse(TaKaRa,日本);DNA 凝胶回收试剂盒(DONGSHENG BIOTECH,中国);SYBR Green PCR Master Mix(TOYOBO,中国);金黄色葡萄球菌 ATCC6538,(鲁微科技有限公司,中国);乙型溶血性血链球菌 ATCC21059,(鲁微科技有限公司,中国);大肠杆菌CMCC(B)44102,(鲁微科技有限公司,中国);台式离心机(中佳,中国);PCR 仪(黑马,中国);倒置光学显微镜(Olympus,日本);紫外分光光度计 ND-1000(NanoDrop,美国);DNA 电泳仪(Tanon,中国);凝胶成像系统(培清科技,中国);荧光定量 PCR 仪(ABI,美国)。
1.2.1 MC3T3-E1细胞培养
将MC3T3-E1细胞培养于DMEM高糖培养基中(含质量分数10%胎牛血清),培养条件为标准条件(体积分数5%CO2、37 ℃、饱和湿度),培养至细胞生长密度达80%时,胰蛋白酶消化并进行传代,取生长状况良好、处于对数生长期细胞进行后续实验。
1.2.2 黄芩溶液配置与作用浓度筛选
配置100 mmol/L 的黄芩素母液并使用0.22 μmol/L 无菌过滤器过滤,根据实验所需DMEM高糖培养基调配成所需浓度。CCK-8法对本次实验选取浓度进行预实验,制备含黄芩素 0(空白对照) 、50、 100、150、200 μmol/L 的培养基,培养 0、1、7、14 d后测定OD值,每组浓度重复3次,检测细胞增殖情况。
1.2.3 实验分组
根据预实验所得出的半数抑制浓度(IC50)为24 μmol/L 作基础,按 100%、75%、50%、25%的比例作梯度细化为后续实验的药物浓度。实验组更换为浓度分别为 6、12、18、24 μmol/L的黄芩素条件培养基[含质量分数10%FBS,青霉素与链霉素双抗(青霉素:10 000 U/mL,链霉素:10 000 µg/mL)],CCK-8实验与成骨基因表达实验对照组更换为α-MEM完全培养基[含质量分数10%FBS,青霉素与链霉素双抗(青霉素:10 000 U/mL,链霉素:10 000 µg/mL)];ALP活性测定实验对照组更换为DMSO完全培养基[含质量分数10%FBS,青霉素与链霉素双抗(青霉素:10 000 U/mL,链霉素:10 000 µg/mL)]。
1.2.4 CCK-8检测
MC3T3-E1以5×104个/mL接种于每孔100 μL的96孔板,每组5个复孔,分别培养1、3、7 d时加入CCK-8试剂检测吸光度值,计算各组细胞相对增殖率与细胞毒性评价。相对增殖率=黄芩素组吸光度均值/阴性对照组吸光度均值×100%。细胞毒性评价标准参考文献中以相对增殖率为评价标准,评级为1~2代表药物有较好安全性(
1.2.5 碱性磷酸酶活性检测
各组细胞分别诱导0、7、14 d后,去除原培养液,PBS冲洗后吸干,每孔加入 500 μL 体积分数0.1%Triton X-100,4 ℃过夜。细胞完全裂解后取细胞裂解液转移至96孔板,每组5个复孔,按碱性磷酸酶(alkaline phosphatase, ALP)试剂盒说明操作,酶标仪测定520 nm处各孔的吸光度值。
1.2.6 成骨相关基因表达检测
各组细胞分别诱导0、7、14 d后,用TRIzol法提取细胞中RNA,艾本德核酸蛋白测定仪上测定吸光度值,确定RNA纯度,RNA电泳测定RNA完整性后反转录成cDNA,RT-PCR检测相关基因表达量。设置内参基因GAPDH-141bp,实时荧光定量PCR检测各组细胞在不同时间Runt相关转录因子2(runt-related transcription factor 2, RunX2)、骨形态发生蛋白 2 (bone morphogenetic protein 2 , BMP-2) 、成骨细胞 (Osterix)的基因表达。引物序列见
Gene | Sequences(5’→3’) |
---|---|
GAPDH | F: GGCCTCCAAGGAGTAAGAAA |
R: GCCCCTCCTGTTATTATGG | |
RUNX2 | F1:TGCCCAGTGAGTAACAGAAAGAC |
R1: CTCCTCCCTTCTCAACCTCTAA | |
Osterix | F1: CCCAACTGTCAGGAGCTAGA |
R1: CAGAGCGAGTGAACCTCTTG | |
BMP2 | F1: AGTCAGTGGGAGAGCTTCGA |
R1: CTTGGAGACACCTGGGTTCT |
1.2.7 抑菌检测
将大肠杆菌、金黄色葡萄球菌、血链球菌接种于肉汤琼脂基斜面,37 ℃培养增殖,稀释成细菌悬液(1×105~1×108) CFU/mL备用。取0、6、12、18、24 μmol/L 黄芩素浸泡滤纸,烘干后放置于涂布实验菌液的琼脂平板上,每组浓度重复测试3次,(35~37) ℃孵育(18~24) h后除测量抑菌圈直径。
采用SPSS23.0进行统计分析,所有数据以例数(n)以及均值±标准差(
CCK-8法比较不同浓度黄芩素对MT3TE-E1细胞增殖的影响,对比各组吸光度值(
Group | 1 d | 3 d | 7 d |
---|---|---|---|
Control | 0.720 2±0.011 8 | 2.375 4±0.014 0 | 2.984 8±0.022 8 |
6 μmol/L | 0.649 2±0.006 1∗ | 1.397 0±0.044 0∗ | 2.320 6±0.037 8∗ |
12 μmol/L | 0.624 0±0.014 2∗ | 1.273 8±0.020 0∗ | 2.097 2±0.048 3∗ |
18 μmol/L | 0.642 8±0.004 3∗ | 1.258 8±0.033 4∗ | 2.141 4±0.006 2∗ |
24 μmol/L | 0.619 6±0.008 6∗ | 1.191 6±0.046 6∗ | 1.755 4±0.060 8∗ |
∗: P<0.05, compare with the control group(all P<0.001). By analysis of variance, there were statistically significant difference between the five groups (day 1: F=88.90, P<0.000 1; day 3: F=1 691.03, P<0.000 1; day 7: F=779.00, P<0.000 1).
图1 不同浓度黄芩素对MC3T3-E1 细胞增殖的影响
Fig. 1 Effects of different concentrations of baicalein on the proliferation of MC3T3-E1 cells
Group | 1 d | 3 d | 7 d | |||
---|---|---|---|---|---|---|
RGR/% | Cytotoxicity | RGR/% | Cytotoxicity | RGR/% | Cytotoxicity | |
0 μmol/L | 100 | 1 | 100 | 1 | 100 | 1 |
6 μmol/L | 90.14 | 1 | 59.70 | 2 | 77.75 | 1 |
12 μmol/L | 86.60 | 1 | 53.62 | 2 | 70.26 | 2 |
18 μmol/L | 89.30 | 1 | 53 | 2 | 71.85 | 2 |
24 μmol/L | 86.03 | 1 | 50.16 | 2 | 58.81 | 2 |
在不同浓度黄芩素下,成骨诱导黄芩素0、7、14 d,评价MC3T3-E1细胞早期成骨分化。结果表明,7、14 d时,黄芩素组的吸光度值均相较对照组显著增高,且呈剂量依赖性(
Group | 0 d | 7 d | 14 d |
---|---|---|---|
0 μmol/L | 0.019 6±0.010 4 | 0.048 3±0.002 9 | 0.094 0±0.020 6 |
6 mol/L | 0.023 4±0.012 5 | 0.070 0±0.007 3∗ | 0.120 9±0.016 7∗ |
12 mol/L | 0.024 8±0.017 2 | 0.080 0±0.014 0∗ | 0.146 6±0.006 1∗ |
18 mol/L | 0.028 0±0.021 2 | 0.100 0±0.008 7∗ | 0.170 0±0.009 4∗ |
∗: P<0.05, compare with the control group (except for the 6 μmol/L group on day 7 P=0.002, and the 6 μmol/L group on day 14 P=0.009, all the other groups were P<0.001 compared with the control group). By analysis of variance,there were statistically significant difference between the four groups in day seven and day fourtine (day 0: F=0.246, P=0.863; day 7: F=28.93, P<0.000 1; day 14: F=26.32, P<0.000 1).
结果可见对照组与各黄芩素组均下调Runx2基因表达水平,且呈浓度依赖性,下调水平与实验时间和实验浓度呈负相关。第14天时,18 μmol/L黄芩素组Runx2基因表达水平较第0天下调将近50 %(
图2 Runx2 mRNA 在 MC3T3-E1 成骨细胞中的表达情况
Fig. 2 The expression of Runx2 mRNA in MC3T3-E1 osteoblasts
∗: P<0.05,compare with the control group (except for the 6μmol/L group on day 0 P=0.002, all the other groups were P<0.001 compared with the control group). By analysis of variance,there were statistically significant difference between the four groups in these three time (day 0: F=42.78, P<0.000 1; day7: F=74.122, P<0.000 1; day14: F=53.82, P<0.001).
图3 BMP2 mRNA 在 MC3T3-E1 成骨细胞中的表达情况
Fig. 3 The expression of BMP2 mRNA in MC3T3-E1 osteoblasts
∗: P<0.05,compare with the control group (in day 0, 6 μmol/L P=0.026; 12 μmol/L P=0.001; 18 μmol/L P<0.001; in day 7, 6 μmol/L P=0.002. In day 14, 6 μmol/L and 12 μmol/L P<0.001; 18 μmol/L P=0.001). By analysis of variance,there were statistically significant difference between the four groups in day0 and day 14 (day 0: F=1 150.69, P<0.000 1; day 7: F=11.10, P=0.05; day14: F=650.90, P<0.001).
图4 Osterix mRNA 在 MC3T3-E1 成骨细胞中的表达情况
Fig. 4 The expression of Osterix mRNA in MC3T3-E1 osteoblasts
∗: P<0.05, compare with the control group (in day 0, 18 μmol/L P=0.05; in day 7, 12 μmol/L and 18 μmol/L P<0.001; in day 14, 6 μmol/L P=0.009, 12 μmol/L P=0.013, 18 μmol/L P<0.001). By analysis of variance, there were statistically significant difference between the four groups in these three time (day 0: F=7.361, P=0.014; day 7: F=17.12, P=0.012; day 14: F=14.277, P=0.002).
K-B 纸片法将0、6、12、18、24 μmol/L 黄芩素作用大肠杆菌、金黄色葡萄球菌、血链球菌 24 h后观察发现,金黄色葡萄球菌和血链球菌在 12 μmol/L黄芩素组开始出现抑菌圈并表现出抑菌效能,抑菌圈的大小和黄芩素浓度呈正相关(表
Group | Bacteriostatic circle diameter/mm | Average bacteriostatic circle diameter/mm | Sensitivity | ||
---|---|---|---|---|---|
1 | 2 | 3 | |||
0 μmol/L | 7 | 8 | 7.5 | 7.5 | - |
6 μmol/L | 8 | 7.5 | 7 | 7.5 | - |
12 μmol/L | 7.5 | 7 | 7 | 7.17 | - |
18 μmol/L | 7 | 7 | 7 | 7 | - |
24 μmol/L | 6 | 8.5 | 7 | 7.17 | - |
“-”insensitive; “+”hyposensitivity; “++”mesosensitivity; “+++”hypersensitivity.
Group | Bacteriostatic circle diameter/mm | Average bacteriostatic circle diameter/mm | Sensitivity | ||
---|---|---|---|---|---|
1 | 2 | 3 | |||
0 μmol/L | 7 | 6.5 | 7.5 | 7 | - |
6 μmol/L | 9.5 | 7 | 7.5 | 8 | + |
12 μmol/L | 15 | 11 | 10 | 12 | + |
18 μmol/L | 16.5 | 17 | 15.5 | 16.33 | ++ |
24 μmol/L | 21.5 | 15.5 | 18 | 18.33 | ++ |
“-”insensitive; “+”hyposensitivity; “++”mesosensitivity; “+++”hypersensitivity.
Group | Bacteriostatic circle diameter/mm | Average bacteriostatic circle diameter/mm | Sensitivity | ||
---|---|---|---|---|---|
1 | 2 | 3 | |||
0 μmol/L | 9 | 6.5 | 8 | 7.83 | - |
6 μmol/L | 9 | 8 | 11.5 | 9.5 | + |
12 μmol/L | 14 | 14 | 13.5 | 13.83 | + |
18 μmol/L | 14.5 | 15.5 | 15.5 | 15.17 | ++ |
24 μmol/L | 20 | 22 | 22.5 | 21.5 | +++ |
“-”insensitive; “+”hyposensitivity; “++”mesosensitivity; “+++”hypersensitivity.
图5 黄芩素作用下大肠杆菌的体外抑菌圈直径
Fig.5 In vitro bacteriostatic circle diameter of Escherichia Coli under the action of baicalein
图6 黄芩素作用下血链球菌的体外抑菌圈直径
Fig. 6 In vitro bacteriostatic circle diameter of Streptococcus Sanguis under the action of baicalein
图7 黄芩素作用下金黄色葡萄球菌的体外抑菌圈直径
Fig. 7 Diameter of bacteriostatic circle of Staphylococcus Aureus in vitro under the action of baicalein
牙周炎、种植体周围炎等疾病都与牙槽骨长期处于慢性炎症状态,导致牙槽骨内骨重建平衡失调相关。黄芩素提取于黄芩类植物根部,已在中医学领域广泛应用多年。前期研究中已有多名学者证实黄芩素可减轻大鼠实验性牙周炎的炎症,降低牙槽骨的吸收以及牙龈纤维的破坏,也有研究证实在临床进行根面平整术时联用黄芩素营养剂,术后患者患处炎症消退情况、探诊出血情况、牙槽骨恢复情况均优于单纯进行根面平整治疗患者,提示黄芩素在临床治疗牙周支持组织疾病时在菌斑炎症的控制、牙槽骨再生治疗方面的优势,在口腔各类炎症性疾病与骨组织工程均有较好的研究前景[
为研究黄芩素对MC3T3-E1成骨分化的影响,我们检验了不同浓度黄芩素培养下ALP的蛋白量。ALP可增加无机磷酸盐的沉积率,促进矿化,并降低细胞外焦磷酸盐浓度,从而促进细胞的成熟与钙化,是最广泛认可的早期成骨活性标志物[
成骨的过程还需各种因子参与,其中,BMP2具有诱导未分化间充质干细胞向成软骨细胞和成骨细胞定向分化与增殖能力,在骨骼发育、最终的骨形成与骨修复都有至关重要的作用[
口腔是连接外消化道与呼吸道的重要场所,微生物多样性在全身各系统位居第二,其微生物群落在口腔乃至全身健康都发挥巨大作用[
本实验将黄芩素的成骨作用与抑制作用结合,期望探索出既能高效抗菌又能促进成骨的适宜剂量。综合考虑不同浓度的黄芩素对成骨以及抑菌方面同时作用的效果,我们认为18 μmol/L的黄芩素可在保证MC3T3-E1细胞正常增殖的同时,有效的促进细胞的成骨分化与抑制口腔常见菌金黄色葡萄球菌、血链球菌的增殖,为黄芩素在牙周炎、种植体周围炎等疾病治疗的应用提供了一定的理论依据。然而本研究还存在一些不足之处。本实验仅进行了体外细胞实验,缺乏在牙周炎、种植体周围炎细胞模型及体内模型实验的进一步验证,我们将在后续的实验中进一步验证。此外,黄芩素对MC3T3-E1产生的分子机制仍需进一步明确。
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