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【摘要】 ：［目的］考察丹参酮ⅡA(TSⅡA)对白细胞介素-1β(IL-1β)诱导的兔纤维环细胞能量代谢障碍的保护作用。［方法］藻酸盐串珠立体培养兔纤维环细胞，将细胞分为7组，在培养过程中加入不同浓度的药物：A组为空白对照不加入药物，B组加入4 μg／ml TSⅡA，C组加入10ng／ml IL-1β，D～G组在给予10 ng／mlIL-1β同时分别加入0．5、1、2和4 μg／ml TSⅡA。于培养3 d后行Na+-K+-ATP酶活性检测、，琥珀酸脱氢酶活性检测、MTT法细胞增殖情况检测以及细胞凋亡的流式细胞仪检测。［结果］G组Na+-K+-ATP酶活性(3.23±0.28U/mgprot)较C组(1.118±0.15U／mgprot)明显增高(P<0.01)，与A组接近(3.57±0.15 U／mgprot)(P>0.05)。G组琥珀酸脱氢酶活性(12.48±0.97U／mgprot)较c组(3.03±0.60 U／mgprot)明显增高(P<0.01)，与A组接近(14.24±1.56 U／mgprot)(P>0.05)。G组MTT试验吸光度(0.77±0.06)较C组(0.31±0.07)明显增高(P＜0.01)，随着TSⅡA浓度的升高，D～G组吸光度随着TsIIA上升而上升。G组细胞死亡细胞比例和凋亡细胞比例分别为21.08±1.46%和8.99±0.33%，均显著低c组(43.11±2.7，P<0.01和11.71±0.32，P<0.01)。［结论］TSIIA能够减轻IL-1β对纤维环细胞能量代谢的抑制作用，从而改善纤维环细胞的增殖、死亡及凋亡。
【关键词】 丹参酮ⅡA 椎间盘 白细胞介素-1β Na+-K+1-ATP酶 琥珀酸脱氢酶
Intervertebral disc degeneration is a common disease. Its mechanism is closely related to obstruction of energy metabolism of intervertebral disc cells［1］. Tanshinone IIA, one of the main active constituents of Salvia Miltiorrhiza, has various effects including the antioxidant effects, scavenging oxygen free radicals, protecting the mitochondrial membrane, and promoting cell proliferation［2］. The purposes of this study are to investigate the protective effects of TSⅡA for proliferation, energy metabolism, and apoptosis of rabbit annulus fibrosus cell and explore the possibilities of treatment for disc degeneration using TSⅡA.
1 Materials and methods
1.1 Materials and animals: TSⅡA standard preparation was purchased from the National Institute for the control of pharmaceutical and biological products; Fetal bovine serum, DMEM-F12 culture medium and collagenase II from Gibco Co. (USA); MTT kit from Lingfei Company of Science and Technology (Wuhan); pancreatin, DMSO, interleukin-1β (IL-1β), alginate from Sigma Co. (USA); the ATPase test kit and the succinate dehydrogenase (SDH) test kit from Jiancheng Biological Company of Science and Technology (Nanjin); BCA quantitative test for protein kit (Wuhan Boster Biological Technology Co. Ltd.), annexin V-FITC kit (Jingmei Biotech Co. Ltd.), Ordinary Japanese rabbit was provided by animals center of Tongji medical college of Huazhong University of Science and Technology.
1.2 Experimental methods
1.2.1 Isolation culture of rabbit annulus fibrosus (AF) cells: Back skins was incised from posterior in sterile environment and exposed lumbar in paragraph 1 to 6 after killed rabbit by venous air embolism, and then take out the lumbar which has exposed paragraph. After that stripping the intervertebral disc, pulposus and non-annulus fibrosus tissue were removed. Annulus fibrosus was cut into the size of the fragments of about 1 mm3 and 0.25% pancreatin was added to digest these tissues in 37℃ for 15 minutes. Then, PBS was used to remove pancreatin and 0.2% collagenase II was added for further digestion in 37℃ for 4 hours. A 200 μm mesh nylon net filter was applied after tissue digestion complete, The filtrate was collected and washed by PBS and centrifugated the filtrate for three times. Finally, 1.2% alginate solution of 2 - 3 ml was added into a suspension (cell concentration of about 1×106/ml）for the production of alginate beads.
1.2.2 Preparation of alginate beads: 5 ml of 102 mmol/L calcium chloride was added into each well of a 6-wells plate and then the alginate suspension was added into each well (30 drops of each well). Standing for 10 minutes, alginate beads were formed. Then calcium chloride solution was aspirated by using a dropper. The beads were washed once by DMEM medium after washed with normal saline for 3 times. Finally, 3 ml of 20% fetal bovine serum of DMEM medium was added and to observe the culture incubator for a week, the next day to replace the culture medium.
1.2.3 Preparation of TS ⅡA solution: Weighing TSⅡA powder 10 mg and adding DMSO 2 ml to dissolve the TSⅡA powder. After TSⅡA completely dissolved, the solution was diluted by 20ml culture medium of DMEM-F12 make the DMSO final concentration below 0.1%. After filter sterilization by 0.22 μm sterile filter preserve in 4 ℃ in darkness.
1.3 Grouping: Rabbit annulus fibrosus (AF) cells were divided into 7 groups according to the finding by Baoqi Wang ［2］ relevant research data, etc. Group A (as normal control，without drug intervention), group B (TSⅡA 4 μg/ml), group C (IL-1β 10ng/ml), group D (IL-1β 10ng/ml + TSⅡA 0.5μg/ml), group E (IL-1β 10ng/ml+TSⅡA 1μg/ml), group F (IL-1β 10ng/ml+TSⅡA 2μg/ml), group G (IL-1β 10ng/ml+TSⅡA 4μg/ml), continuous drug intervention for 3 days.
1.4 Detection indicator: Culture medium was removel and alginate beads were washed using physiological saline after 3 consecutive days of drug intervention. Then, alginate beads were dissolved by adding 2 ml lysis liquid (contain 3.5mmol/l citrate sodium and 150 mmol/l sodium chloride) and centrifugated (1800rpm/minute) for 10 minutes to collect cells. Afterwards, cells of each group was detected and detection indicators including ATP assay, succinate dehydrogenase (SDH) activity assay and MTT cell proliferation assay, detection of each specimen were repeated 3 times. MTT testing five-hole, according to the OD value in wavelength of 570 nm to speculate that the number of live cells.
1.5 Apoptosis detection: The proportion of apoptosis and death cells were tested by Annexin V-PI staining and flow cytometry. Cells were centrifuged after washing for 2 times using pre-cooled at 4 ℃ in phosphate buffer（PBS), and then cells were prepared into 1 × 106/ml suspension by binding buffer. At room temperature, the cell suspensions were incubated with Annexin V reagent for 15 minutes in darkness. Then the proportion of apoptosis and cell death of Annexin V+/ PI-cells were tested by flow cytometry within 1 hour.
1.6 Statistical methods: The results were showed through ±s, apply SPSS12.0 software using one-way ANOVA, compared with group A using Dunnett-t test, Data between each group compared using SNK 22 Test, Repeat three times with flow cytometry test, results analysis of variance.
2 Results
2.1 Na+-K+-ATPase and SDH activity
The Na+-K+-ATPase and SDH activity (U/mgprot) in each group (A-G) was showed in table 1. In group B, the activity of two enzymes has no significant change than group A. In group C, the activity of Na+-K+-ATPase and SDH were significantly lower than that of group A (P<0.01,P<0.01). In groups D～G, with the TSⅡA higher concentration of both enzyme activities were significantly enhanced, G unit Na+-K+-ATPase activity than the group C for nearly 3 times(P<0.01) and the SDH activity is more than 3 times higher than group C(P<0.01).
2.2 MTT test
Absorbance in each group (A-G; OD value) was showed in table 1. Group B than A group absorbance increase, but there is no statistical significance (P>0.05),group C than group A was significantly decreased (P<0.01). Absorbance in group D is significantly higher than group C (P<0.01), with the concentration of TS Ⅱ A higher absorbance was gradually increased. Absorbances in group E, F, G are close to group A. Absorbance in group G is 2.46 times than group C (P<0.01).Table 1 the test result of each groupNa+-K+-ATPase（略）
2.3 The proportion of apoptotic and death cells
The proportion of apoptotic and death cells in each group (A-G) (%) was showed in Table 1 and Fig 1. In group B, the proportion of death cells were lower than group A, but there is no statistical significance (F=5.73, P=0.075). Group C was significantly increased than group A (F=239.046, P<0.01). Group D was significantly lower than that of group C (F=22.351, P<0.01). As the concentration of TSⅡ A increased, the proportion of death cells in groups E, F and G were further declined. The proportion of death cells in group G dropped by about 52% compared to group C (F=153.87, P<0.01).
The proportion of apoptotic in group B was significantly decreased than group A (F=980.761, P<0.01), C Group was higher than group A (F=0.093, P<0.01). In group D, the proportion of apoptotic was lower than group C (F=34.124, P<0.01). In groups E and F, the proportion of apoptotic were higher than group D (F=5.485, P =0.081; F=8.137, P=0.046), and group F has statistically difference. Group G was lower than groups C or D (F=17.997, P=0.013; F=107.677, P<0.01), and even lower than group A (F=19.367, P=0.012).
3 Discussion
Excessive secretion of inflammatory factors is an important mechanism in the course of intervertebral disc degeneration. IL-1β is an inflammatory factor with multiple biological effects. The increase in secretion of IL-1β was induced by external factors such as pressure. This can not only directly activate the disc cellular apoptosis process and also significantly enhance the expression of inflammatory factors such as the nitric oxide and tumor necrosis factor, interfere with cellular energy metabolism and induce cell apoptosis［3］. Therefore, in this study we chose IL-1β to induce damage of annulus fibrosus cell. Modern scientific research demonstates that salvia miltiorrhiza can promote energy metabolism, speed up the synthesis of ATP and inhibit inflammation［4］. Therefore, we chose TSⅡA, a major component of salvia miltiorrhiza, to inhibit effects of IL-1β on cell energy metabolism, proliferation, and apoptosis. Fig.1the proportion of apoptotic and death cells Cell apoptosis in groups D-G showed that 0.5μg/ml TSⅡA could significantly reduced the proportion of apoptosis and cell death. Accompanying the increment in the concentration of TSⅡA, a slight increase in apoptosis rate of annulus fibrosus cells was observed, but the death of cells in the gradual reduction. Until treatment of 4μg/ml TSⅡA, the proportion of death cells was still significantly higher than the blank control group. The ratio of apoptotic cells even lower than the blank control groupMitochondrial energy metabolism has a direct relationship with functions of four respiratory chain complex, the cells intake of glucose and oxygen, and Na+-K+-ATPase activity. Na+-K+-ATPase not only can take the initiative to transmembrane Na+-K+ ion, but through the cytoplasmic signal transduction pathways within the mitochondria to activate protein kinase C (PKC) to inhibiting mitochondrial membrane permeability to avoid cell death or apoptosis. IL-1β not only can directly inhibit Na+-K+-ATPase activity, but also can indirectly inhibit Na+-K+-ATPase activity by affecting mitochondrial energy metabolism of cells leading to ATP production decrease［5］. The results of this study show that, 1μg/ml TSⅡA can significantly alleviate that inhibition of IL-1β on the annulus fibrosus cells in Na+-K+-ATPase, 4 μg/ml TSⅡA allows Na+-K+-ATPase maintained at close to control group level. This role contributes to the maintenance of mitochondrial membrane stability and cell energy metabolism to avoid apoptosis. In addition, TSⅡA promoted the expression of PKC［6］ and inhibited impact of IL-1β on mitochondria.
SDH is a combination enzyme of mitochondrial inner membrane, one of linking oxidative phosphorylation and electron transport hub. The activity is an important indicator to evaluate the operation of the degree of the TCA cycle. During the mitochondrial energy metabolism and electron transfer process, a small amount of leakage of oxygen ions is produced to form clusters of reactive oxygen species (ROS), inhibit cell metabolism. Excessive ROS can even lead to cell apoptosis. In normal circumstances, the antioxidant enzymes in cells such as superoxide dismutase (SOD) can fight against the role of ROS. However, IL-1β inhibit the generation of antioxidant enzymes which resulted in a marked increase in ROS, caused mitochondrial dysfunction, and can also inhibit the activity of SDH and reduce the production of ATP［7、8］. This study shows that, TSⅡA can be to reduce IL-1β on inhibition of SDH activity through the dose-effectiveness patterns, consistent with the findings of Zhou et al［9］. Therefore, TSⅡA may contribute to the stability of mitochondrial membrane of annulus fibrosus cells, to increase their ATP production and reduce the release of ROS, thus improving the biological behavior of annulus fibrosus cells.
This study shows that simply adding TSⅡA does not make the annulus fibrosus cell proliferation significantly higher than the blank control group, but 0.5μg/ml TS Ⅱ A could significant improve the inhibition that IL-1β imposed on the annulus fibrosus cells. However, increasing TSⅡA concentration can not completely reverse the inhibition, which may be related to various biological effect of IL-1βthat can not be completely inhibited.
After treatment of TSⅡA, the proportion of apoptosis and death of annulus fibrosus cells decreased significantly than that of the control group. TSⅡA can help annulus fibrosus cells resist to various injuries in the process of experiment. Cell apoptosis in groups D-G showed that 0.5μg/ml TSⅡA can be significantly reduced the proportion of apoptosis and cell death. Accompanying the increment in the concentration of TSⅡA, a slight increase in apoptosis rate of annulus fibrosus cells was observed, but the proportion of death cells in each group was significantly decreased at this time. The total number of the proportion of apoptosis and death of annulus fibrosus cells in each group maintained downward trend with the increase in concentration of TSⅡA. Until treatment of 4μg/ml TSⅡA, the proportion of death cells was still significantly higher than the blank control group. The ratio of apoptotic cells even lower than the blank control group. The results suggest that TSⅡA has a strong effect in inhibition apoptosis of annulus fibrosus cell. However, it does not completely prevent the effect of IL-1β. Combination of apoptosis and proliferation can be found that the role of TSⅡA, in a larger dose, on the one hand, the apoptosis decreased significantly. On the other hand, cell proliferation can be maintained at a high level, it's suggesting that TSⅡ A may be able to help reverse the downward trend in the number of annulus fibrosus cell in the process of disc degenerations, which is essential to treat disc degeneration.
In conclusion, TSⅡA can improve energy metabolism of the annulus fibrosus cells and help annulus fibrosus cells to resist the impact of IL-1β on cell energy metabolism, proliferation, apoptosis and biological behavior. Theoretically, TSⅡA has potential to treatment for disc degeneration. However, before TSⅡA is used for clinical application, many problems need to be solved. For example, can TSⅡA go through the cartilage end plate barriers; the concentration formed in local disc was not known; In addition, the effect of long-term medication is not clear. Thus, in-depth study of Chinese medicine for the treatment of disc degeneration will help us to solve these problems to obtain a more rational and effective treatment for disc degeneration.
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作者单位：华中科技大学同济医学院附属协和医院骨科，武汉 430022