Inhibitory effect of Sumu(Lignum Sappan)plus Fuzi(Radix Aconiti LateralisPraeparata)on a lung carcinoma model

INTRODUCTION

Malignant tumors are severe diseases that seriously threaten the health and life of humans.There is a considerable amount of literature demonstrating that radiochemotherapy combined with Chinese medicine can relieve clinical symptoms,maximize quality of life,and prolong survival.1MicroRNAs are widely regarded as biological diagnostic and prognostic markers of tumors in clinical settings.2Recently,Chinese medicine was shown to inhibit some tumors by upregulating the expression of microRNAs.3

Scholars hold different views on the application of drugs for invigorating blood circulation and eliminating stasis,as commonly applied in oncotherapy.Some believe that multiple targets can lead to inhibition of the mechanism for the initiation,progression,and metastasis of tumors.4-7Research has shown that heparin,which has similar effects to drugs that invigorate blood circulation and eliminate stasis,has anti-neovascularization and tumor growth inhibition effects.It has also been reported that heparin can reduce the adhesion of malignant cells,resulting in reduced tumor invasion and metastasis.8-11

18 Han R.Research and development of anticancer drugs and experimental techniques.Beijing:United Press of Beijing Medical University and Peking Union Medical College,1997:370.

Lung cancer is included in the top five cancers in all countries,and ranks highly in terms of mortality worldwide.15Through animal experiments,Tianet al16,17found that drugs for invigorating blood circulation and eliminating stasis,such as Shuizhi(Hirudo),Chuanxiong(Rhizoma Chuanxiong),Jixueteng(Caulis Spatholobi),and Sumu(Lignum Sappan),can inhibit the growth and metastasis of tumors.The present study assessed the inhibitory effect of Sumu(Lignum Sappan)plus Fuzi(Radix Aconiti Lateralis Praeparata)(SF)at different concentrations on the growth and metastasis of Lewis lung carcinoma(LLC),with the aim of determining a better combination of herbal ingredients.

METHODS

Experimental model and main outcome measures

Clean inbred strain C57BL/6 male mice aged 6-8 weeks and weighing 18-22 g were provided by the Laboratory Animal Research Institute of the China Academy of Medical Science[License:SCXK (Beijing)2016-0004].An LLC cell line,with a 100%growth rate of tumors after transplantation,was provided by the National Infrastructure of Cell Line Resource(Beijing,China).The LLC cells were thawed from liquid nitrogen storage and subcutaneously inoculated into the right forelimb armpit of mice.After two passages of cultured-cells,the lungs of the mice were removed,ground,filtered,and trypsinized under aseptic conditions,and a single-cell suspension of lung tissue cells was created on day 20.After dilution,trypan blue assays confirmed that the survival rate of LLC cells was＞95%.Gavaged mice were subcutaneously inoculated into the right forelimb armpit with LLC cells resuspended at 1.0×107cells/mL(0.2 mL/mouse).18Tumor size and body weight were measured and recorded every day.Mice were euthanized by dislocation of the cervical vertebra on day 21.All mice were drawn blood from eyeball and killed.Tumor tissue was obtained under the skin of the right forelimb armpit for tumor weight measurement.Lung tissue was extracted to observe lung metastasis,and expression of sP-Selectin/CD62P and vascular endothelial growth factor C(VEGFC)in tumor tissue was investigated.Serum was separated by centrifugation at 1071×gat 4℃,and stored at－80℃until analysis.sP-Selectin/CD62P was measured by enzyme-linked immunosorbent assay(ELISA)within 1 week using an sP-Selectin/CD62P Detection Kit(R&D Systems,Minneapolis,MN,USA).All experimental procedures in this study were approved by the Experimental Animal Ethics Committee of Beijing University of Chinese Medicine.

除了向身边的同事学习，如果条件允许还应该多向区域内的专家积极学习，笔者有幸在参加工作的第二年参加了“东营市胜利教育管理中心初中数学高级研修班”和“东营市孙庆民名师工作室”的学习，结识了山东省正高级教师孙庆民，一路走来，受益匪浅，收获颇多.

Traditional Chinese medicine compound

For SF decoction(ratio of 1∶1)administration to LLC model mice,the low dose was approximately 10×the adult human(60 kg)clinical dosage per kilogram(prepared at 0.3 g/mL),the moderate dosage was 2×higher than the low dose,and the high dosage was 3×higher than the low dose.An adult intake of Sumu(Lignum Sappan)is 10 g/d.The Chinese herbs were provided by Beijing Tongrentang(Beijing,China).

Grouping and administration

A total of 52 mice were randomly divided into four groups of 13 mice:control group,low-dose group,moderate-dosage group,and high-dosage group.Each mouse was weighed at 24 h after inoculation.The SF-treated micewereintragastricallyadministered0.2mL of SF decoction every day,while the control mice were administered distilled water.

增强班级的凝聚力，在农村来说，说起来容易，但作起来难．只要我们大家能勇于创新，勇于研究，不断地发现困难，总结经验，相信会建立起一个生机勃勃的班集体．班集体的凝聚力的强弱则可以反映班风、学风的优劣。一个优秀的有凝聚力的班集体应该是有共同目标、有组织核心、有正确舆论、有旺盛士气的集体。

Inhibition rates

Tumor weights were measured on an electronic balance,and the tumor inhibition rate was calculated by the following formula:Growth inhibition rate=[mean weight of tumor in control group(g)－mean weight of tumor in experimental group(g)]/mean weight of tumor in control group(g)×100%.

Lung metastasis rates

Lung tissues were observed at 24-48 h after fixation with 35%-40%formaldehyde solution.Lung tissue appeared yellow,while metastases appeared white.The size and number of metastases were observed under an anatomical microscope.Tumor metastasis was graded according to size as follows:gradeⅠ,diameter less than 0.5 mm;gradeⅡ,diameter between 0.5 mm and 1 mm;gradeⅢ,diameter between 1 mm and 2 mm;gradeⅣ,diameter over 2 mm.The total metastasis number was calculated by the following formula:19Total metastasis number=gradeⅠ(total number)×1+gradeⅡ (total number)×2+gradeⅢ (total number)×3+gradeⅣ(total number)×4.The metastasis inhibition rate was calculated as follows:Lung metastasis inhibition rate(%)=(number of lung metastases in control group－number of lung metastases in experimental group)/number of lung metastases in control group×100%.

Enzyme-linked immunosorbent assay(ELISA)

The ELISA analyses were performed by specially-assigned persons according to the kit instructions.The data were plotted graphically,with the abscissa representing the concentration of the standard,and the ordinate representing the optical density(OD)value.A calibration curve was established,and the sample contents were determined according to this calibration curve.

Real-time quantitative RT-PCR(qRT-PCR)

The mRNAs isolated from tumor tissues were analyzed by real-time qRT-PCR.For this,total RNA was extracted using TRIzol®Reagent(Life Technologies Corporation,Carlsbad,CA,USA)in accordance with the manufacturer's instructions,and 1 μL of the extract was electrophoresed to detect the RNA concentration.The extracted RNA was reverse-transcribed to cDNA using an M-MLV Reverse Transcriptase cDNA Kit(Invitrogen,Carlsbad,CA,USA).The cDNA was amplified in duplicate using a Real Master Mix(SYBR Green)Kit(NewBioIndustry,Tianjin,China).After an initial denaturation step at 95℃for 5 min,temperature cycling was initiated.Each cycle consisted of denaturation at 95℃for 15 s,annealing at 55℃for 10 s,and elongation at 72℃for 15 s.A total of 45 cycles were performed.Next,1 μL of cDNA from each sample was used as a template for transcription,with amplification using primers for the target gene and β-actin as an endogenous control gene.A dissociation curve for the analysis was performed at 60-95℃.The sequences of the primer pairs were as follows:VEGFC,5'-TTCCTCATACATACCCAAGA-3'and 5'-CACAGTCCCAGAACCACA-3';β-actin,5'-ATATCGCTGCGCTGGTCGTC-3'and 5'-AGGATGGCGTGAGGGAGAGC-3'.

For data normalization,the endogenous β-actin gene was assessed to control for the cDNA input,and the relative units were calculated by the comparative Ct method.All real-time qRT-PCR experiments were repeated three times,and the results were presented as mean ratios±standard error of the mean.

Statistical analysis

The data were presented as mean±standard deviation(±s),and were analyzed using SPSS for Windows Version 16.0(SPSS Inc.,Chicago,IL,USA).Mouse body weights were evaluated by single-factor analysis of variance.The Student-Newman-Keuls(SNK)-qtest and single-factor analysis of variance were applied to tumor weights and expressions of sP-Selectin/CD62P and VEGFC.Inhibition rates were analyzed by the Cochran-Mantel-Haenzel(CMH)χ2test.Differences for all tests were regarded as statistically significant when theP-value from a two-tailed test was less than 0.05.

RESULTS

Mouse body weight

The mean body weights in all groups did not differ significantly(P=0.776).The data for comparisons of mouse body weights among the different groups are shown in Table 1.

Tumor weight and tumor inhibition rate

15 Saika K,Sobue T.Cancer statistics in the world.Gan to Kagaku Ryoho 2013;40(13):2475-2480.

Table 1 Mouse body weights in the four groups(ˉ±s)

Notes:control group:treated with distilled water;low-dose group:treated with SF decoction(0.3 g/mL);moderate-dosage group:treated with SF decoction(0.6 g/mL);high-dosage group:treated with SF decoction(0.9 g/mL).SF:Sumu(Lignum Sappan)plus Fuzi(Radix Aconiti Lateralis Praeparata).There was no significant difference between different group(P=0.776).

nGroup Control Low-dose Moderate-dosage High-dosage 13 13 13 13 Weight(g)25.9±1.5 26.0±1.0 26.0±1.4 25.5±1.3?

Table 2 Tumor weights and inhibition rates in the four groups on day 21(±s)

Notes:control group:treated with distilled water;low-dose group:treated with SF decoction(0.3 g/mL);moderate-dosage group:treated with SF decoction(0.6 g/mL);high-dosage group:treated with SF decoction(0.9 g/mL).SF:Sumu(Lignum Sappan)plus Fuzi(Radix Aconiti Lateralis Praeparata).Compared with control group,aP＜0.05;compared with high-dosage group,bP＜0.05.

nGroup Control Low-dose Moderate-dosage High-dosage 13 13 13 13 Weight(g)0.95±0.13 0.82±0.04ab0.77±0.03ab0.92±0.08 Inhibitory rate(%)-14.10 19.09 2.66

Lung metastasis

No lung metastases were observed on day 21.

Calibration curve and expression of sP-Selectin/CD62P

Known samples were analyzed to establish a calibration curve and regression equation,and the measured values were applied to the equation to obtain the concentrations of sP-Selectin/CD62P in the experimental samples.The values and concentrations are shown in Table 3,the calibration curve of known samples showed good linearity(r2=0.99902)within the linear range(0-20 ng/mL)and the equation is Y=0.03253+0.23899×X－0.00541×X2(Y,OD value;X,concentration of experimental samples).The calibration curve is shown in Figure 1.

The abscissa represents the concentration of the standard,and the ordinate represents the OD value,where OD=1og(1/trans),with"trans"being the transmittance of the detector.There was a significant difference in the expressions of sP-Selectin/CD62P among thedifferent groups(P=0.001).The expression levels of sP-Selectin/CD62P in the low-dose group and moderate-dose group were lower than those in the control group and high-dosage group(bothP＜0.05),suggesting an antitumor effect.There was no significant difference between the high-dosage group and the control group(P=0.678)(Figure 2).

Table 3 Values and concentrations in the known samples

Notes:1-7:7 known samples with different concentration.OD:optical density.

Samples number 1 2 3 4 5 6 7 8 OD value 0.008 0.096 0.169 0.334 0.622 1.140 1.834 2.726 Concentration(ng/mL)0.000 0.310 0.625 1.250 2.500 5.000 10.000 20.000

Figure 1 Calibration curve of known samples OD:optical density.

Figure 2 Expressions of sP-selectin/CD62P in the different groups

Control group treated with distilled water,low-dose treated with Sumu(Lignum Sappan)plus Fuzi(Radix Aconiti Lateralis Praeparata)(SF)decoction(0.3 g/mL),moderate-dose treated with SF decoction(0.6 g/mL),high-dose treated with SF decoction(0.9 g/mL).Data are presented as mean±standard deviation(n=13).Compared with the control(distilled water treated)group,significant differences were found in the low-dose(SF treated)and moderate-dosage(SF treated)(aP＜0.05),while no difference was found in the high-dosage(SF treated)group(bP=0.678).

Figure 3 Dissociation curve of vascular endothelial growth factor C

Figure 4 Amplification plot of vascular endothelial growth factor C mRNA

Figure 5 Expression levels of VEGFC mRNA in the different groups

Significant differences compared with control(distilled water treated)group was designated asaP＜0.05,and with high-dosage asbP＜0.01.VEGFC:vascular endothelial growth factor C.

Expression of VEGFC mRNA

There was a significant difference in the expression levels of VEGFC mRNA among the different groups(P＜0.01).The expression levels of VEGFC mRNA in the low-dose group and moderate-dosage group were lower than those in the control group and high-dosage group,suggesting an antitumor effect.The dissociation curve and amplification plots of VEGFC mRNA are shown in Figures 3 and 4.

Comparisons of the expression levels of VEGFC mRNA among the different groups are shown in Figure 5.

DISCUSSION

This study was aimed to investigate the inhibitory effect of SF administration on a lung carcinoma model established by subcutaneously inoculating LLC cells into C57BL/6 mice.Chinese medicine preparations have been used for cancer treatment，since it has the function of regulatingYinandYang,as well asQiand blood.Researchers have found that antitumor prescriptions supplementingQito invigorate the spleen and warmingYangto dispel cold herbs have better effects than blood-promoting drugs alone.20The Spiritual Pivot of Huangdi's Canon of Medicine21holds that accumulation ofYincold,which can also be described or defined as excessiveYinwithYangdeficiency,is the etiology of malignancy.The Treatise on Causes and Symptoms of Diseases22proposes that deficiency of bothQiand blood,and stagnation ofQi,resulting in accumulation ofYincold.All of these ancient documents provide a theoretical base for warming the interior to dissipate cold for oncotherapy.

It was reported that warming therapy has a curative effect in oncotherapy.23By analyzing relevant literature,researchers found that warming herbs can improve the stability of the tumor microenvironment as well as the genetic stability of mesenchymal stem cells,resulting in inhibition of tumor generation and metastasis.24Some researchers consider that deficiency ofYangleads to decreased immunological activity,which underlies the pathogenicity of tumors.25Phlegm muddy ties is a pathogen from pathological products.The malfunction of the viscera is a key factor leading to the formation of pathogenic phlegm muddy ties.A hypercoagulative state of blood,the result of cold coagulation and phlegm muddy ties,is an essential condition for tumor formation.Heavy warmingYangin palliative treatment can prolong the survival of patients,26despite the presence of tumors.A clinical study showed that warming yang can ameliorate the symptoms of lung cancer and improve quality of life.27Other research showed that Fuzi(Radix Aconiti Lateralis Praeparata),as representativeYangwarming herbs,can alleviate the symptoms ofYangdeficiency syndrome in lung cancer-bearing mice,eventually delaying tumor progression.28In a previous study,we found that SF had a better effect on the growth and metastasis of LLC than Sumu(Lignum Sappan).29

PDCA循环中主要包括四方面的内容，第一计划，第二实施，第三检查，第四处理。其循环理论主要体现在其四个阶段的相互影响、综合推进中，其理论中四个阶段的实施计划与结果是紧密相连的。将其应用到护理管理中，需要护理人员能够在护理的过程中基于患者基本病况提出问题，并寻找解决的途径，其护理实施不再是无目的、无计划性的，而是要能够制定合理的工作计划，并根据自身的护理计划对患者实施护理。采取PDCA管理能够有效提高护理人员工作的有效性、提高其服务的质量，从而使患者获得更为满意的护理体验。

CONCLUSION:SF can inhibit the growth and metastasis of Lewis lung carcinoma.

Even though the death cannot be prevented,we consider blood-activating medicinal substances combined withYangwarming and activating medicinal substances can improve the long-term survival rate of lung cancer.41,42Stagnations ofQiand blood,resulting from accumulation ofYincold,are clinicopathologic parame-ters in tumors,suggesting that a therapeutic regimen ofYangwarming and strengthening is appropriate to promote smooth blood flow.Blood-activating medicinal substances alone may lower body resistance,resulting in tumor cell invasion and metastasis.

In conclusion,SF can inhibit the expression of P-selectin,suggesting an antitumor effect,and this effect is associated with the applied dose.Further studies are needed to observe the long-term survival rate and the optimal dosage for tumor treatment.

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全学科资源服务重点在期刊、图书、专利，辅之以词表、工具书。内容更新速度必须较快，数据较全，否则没有竞争力。

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Interactions between P-selectin and its ligands may be involved in many kinds of physiological and pathological processes,and are of vital importance for the initiation and development of tumors.30,31Reitteret al32found that the levels of sP-selectin were significantly higher in patients with distant metastases than in those without such metastases.Meanwhile,P-selectin loss may be associated with inhibition of melanoma metastasis to the lung.33Deficiency of P-selectin glycoprotein ligand-1,which is upregulated on responding T cells,led to PD-1 downregulation,improved T cell responses,and tumor control.34Some scholars believe that inhibition of P-selectin or competition with ligands may be novel therapeutic strategies for oncotherapy.35,36Yueet al37found that holothurian glycosaminoglycan behaved as a ligand-competitive inhibitor of P-selectin,and disrupted its binding to cell surface receptors that activate downstream regulators of tumor cell migration.Carcinoma growth,infiltration,metastasis,and recurrence depend on the generation of vessels and lymphatic ducts.Inhibition of this vessel and lymphatic duct generation plays an important role against tumor promotion.VEGFC,one of the most widely-studied angiogenesis factors,can regulate the angiogenesis and lymphangiogenesis of tumors.38VEGFC is mainly relevant to primary tumors and lymph-node metastases.39The VEGF levels were elevated in the presence of tumors and decreased following tumor regression,suggesting that VEGF levels could be a predictor for recurrence.High VEGF levels were correlated with poor prognosis and lower survival rates.Therefore,inhibition of VEGFC expression has become a new strategy in oncotherapy to block the tumor angiogenesis signaling pathway.40

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我们看到大多数家庭经过一场场疾病走向阳光和健康，但也有的父母像轩轩的爸爸妈妈那样，在疾病面前自己先变成了弱者，总在心里向疾病求饶，替宝宝“负罪”，失去了一颗平常健康的心态，给宝宝的性格打上一些特殊的烙印：疾病是一个砝码，可以换来我想得到的一切，尤其是有需要就能满足的优越感。因此，我们常听见有的父母说：“宝宝刚刚大病一场，现在变得特别娇气，有点要求不满足，就哭得像个泪人儿似的。”有的父母则说：“生完病，宝宝的脾气特别大，不像以前那样乖，总想迫使大人让着他。”

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安全壳[20-21]是压水堆的关键设备，是反应堆的最后一道安全屏障，其内层钢制安全壳是一种典型的薄壁压力容器，体积大、厚度薄，包括上、下封头与圆柱壳段，其中有许多开孔、接管、贯穿件等。本文以安全壳的圆柱壳段为研究模型，对开孔和补强的圆柱壳进行轴压屈曲试验和非线性有限元分析。缩比实验的设计与实现有助于获得合适的有限元模型。近年来，钢制壳体的屈曲试验得到了广泛的开展[22]，但试验模型较小，与安全壳的径厚比相差甚远，亦缺少人造局部缺陷和补强等因素。

路边的树木窸窸窣窣地抖着叶子，像一群变魔术的孩子，光怪陆离的影子斑驳地投射在平坦的柏油路上。我小心地踏着它们的影子，背上的小书包发出咣当咣当的声音，我幻想我是一列小火车，妈妈的手是火车的动力，只要妈妈在，我就不会在那些红绿灯路口徘徊，而是直奔自己的方向。还记得那些下雨的日子，我趴在爸爸的背上，雨滴敲打小书包，好像催眠曲，往往还在梦乡里嬉戏，就到家了，我就开始生气：这条路怎么这么短。到了下雪的日子，爸爸紧紧地牵着我的手，慢慢地走啊走，不时还会摔一跤，我就开始生气：这条路怎么这么长。

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A significant difference in tumor weights was observed among the groups(P＜0.05).The tumor weights in the low-dose group and moderate-dosage group were lower than those in the control group and high-dosagegroup(bothP＜0.05).Significant differences were not found between the control group and the high-dosage group(P=0.457),or between the low-dose group and the moderate-dosage group(P=0.158).There was no significant difference(P＞0.05)in the tumor inhibition rates among the different groups(Table 2).

一是加快贫困村集体经济发展。大力推进资源开发型、资产盘活型、产业带动型等村级集体经济发展模式，鼓励将符合条件的财政支农资金形成的资产优先折股量化给贫困村集体，确保2018年底贫困村集体经济年收入达到3万元以上。

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Others believe that blood stasis is a state of self protec-tion that can inhibit the growth and metastasis of tumors.12They consider that drugs for invigorating blood circulation and eliminating stasis can stimulate blood circulation,increase tumor cell mobility,and promote neoplasm metastasis,13and this has been confirmed by research.14

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俗话说得好“母猪好好一窝，公猪好好一坡”，这句话说明了种猪在生产中的重要性。一般来说，公猪应选择生长速度快、瘦肉率高、饲料报酬高的品种，如长白公猪、约克公猪、大白猪等。母猪选择繁殖力强、窝产仔多、泌乳力强、护仔性好、生长快、饲料报酬高等优点的母本品种，散养农户一般选择培育品种、土洋杂交品种、地方品种等，专业养殖户一般选择外来品种、配套系品种等，中等规模养殖户一般选择洋三元品种、高效配套品种等，大型养殖场一般选择洋三元、洋五元及高效配套品种等。这样才能保证母猪产下发育优良的仔猪。

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36 Yu Y,Zhou XD,Liu YK.Advances in the research of P-selectin and it's ligands in tumor metastasis.Zhong Guo Ai Zheng Za Zhi 2000;10(5):443-445.

37 Yue Z,Wang A,Zhu Z,et al.Holothurian glycosaminoglycan inhibits metastasisviainhibition of P-selectin in B16F10 melanoma cells.Mol Cell Biochem 2015;410(1-2):143-154.

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科学管理之父泰罗认为，管理就是“确切地知道你要别人去干什么，并使他用最好的方法去干”。[4]在泰罗的眼里，管理就是指挥他人能用其最好的工作方法去工作。20世纪末，“知识管理”首次出现在学术界，不同领域的学者从不同视角给出了一些定义。英国学者Rowley认为，[5]知识管理关注的是在促进实现组织目标观念指导下，如何对组织知识资产进行挖掘与开发。知识管理的对象，既包括外显的、文档化的知识，也包括内隐的、主观化的知识。知识管理的过程包括知识的识别、共享和创造等过程。它要求创建和共享知识体系，培养和促进组织学习。

40 Bueno MJ,Mouron S,Quintela-Fandino M.Personalising and targeting antiangiogenic resistance:a complex and multifactorial approach.Br J Cancer 2017;116(9):1119-1125.

41 Wei YM,Liang J,Zheng XL,et al.Lung-targeting drug delivery system of baicalin-loaded nanoliposomes:development,biodistribution in rabbits,and pharmacodynamics in nude mice bearing orthotopic human lung cancer.Int J Nanomedicine 2017;12:251-261.

42 Yang S,Cui M,Li HY,Zhao YK,Gao YH,Zhu HY.Meta-analysis of the effectiveness of Chinese and Western integrative medicine on medium and advanced lung cancer.Chin J Integr Med 2012;18(11):862-867.