Effect of p27 Gene Combined with Pientzehuang (片仔癀) on Tumor Growth in Osteosarcoma-Bearing Nude Mice

Objective： To observe the effect of p27 gene recombinant adenovirus combined with Chinese medicine Pientzehuang （片仔癀） on the growth of xenografted human osteosarcoma in nude mice. Methods： Tissue transplantation was used to construct the orthotopic model of human osteosarcoma Saos-2 cell in nude mice. Thirty tumor-bearing nude mice were randomly divided into 5 groups with 6 mice in each group： blank control group （model of osteosarcoma）, empty vector group （recombinant adeno-associated virus-multiple cloning site）, Pientzehuang group, p27 gene group and combined treatment group （p27 gene combined with Pientzehuang）. The effect of combined treatment on human osteosarcoma was analyzed through the tumor formation, tumor volume and inhibition rate of tumor growth. The expression of p27 was measured by immunohistochemical staining and Western blot. Results： The orthotopic model of osteosarcoma in nude mice was successfully constructed. The general appearance of tumor-bearing nude mice in Pientzehuang and p27 gene groups was markedly improved compared with the blank control group; and in the combined treatment group it was significantly improved compared with the Pientzehuang and p27 gene groups. The tumor growth in the Pientzehuang and p27 gene groups was significantly inhibited compared with the blank control group （P〈0.05）; while in the combined treatment group it was markedly inhibited compared with the Pientzehuang and p27 gene groups （P〈0.05）. The rates of tumor growth inhibition were 34.1%, 56.5% and 63.8% in the Pientzehuang, p27 gene and combined treatment groups, respectively. Meanwhile, the protein expression of p27 gene in the p27 gene group was significantly increased compared with the blank control group （P〈0.05）; and it was significantly increased in the combined treatment group compared with the p27 gene and Pientzehuang groups （P〈0.05）. Conclusion： p27 gene introduced by adenovirus combined with Pientzehuang can inhibit the growth of human osteosarcoma cell Saos-2 in nude mice.

片仔癀对糖尿病牙周炎大鼠Keap1/Nrf2通路及牙槽骨丢失的影响

目的:探讨片仔癀对糖尿病牙周炎大鼠Kelch样ECH相关蛋白1(Keap1)/核因子红血球相关因子2(Nrf2)通路及牙槽骨丢失的影响。方法:建立糖尿病牙周炎大鼠模型,随机分为模型组、牙周基础治疗组、片仔癀低剂量(0.75 g/kg)+牙周基础治疗组、片仔癀中剂量(1.5 g/kg)+牙周基础治疗组、片仔癀高剂量(3.0 g/kg)+牙周基础治疗组,每组12只,另取12只设为对照组。分组治疗后,以牙龈指数评估各组大鼠牙周组织炎症情况;通过断层扫描检测各组大鼠牙槽骨吸收值;通过HE染色检测各组大鼠牙周组织病理变化;通过试剂盒检测各组大鼠牙周组织超氧化物歧化酶(SOD)和丙二醛(MDA)水平;通过ELISA法检测各组大鼠血清中IL-6和TNF-α水平;通过Western blot检测各组大鼠牙周组织Keap1/Nrf2通路相关蛋白(Keap1、Nrf2)表达水平。结果:与对照组相比,模型组大鼠牙周组织牙周膜结构紊乱,纤维束排列异常,出现断裂,且伴随炎症细胞大量浸润等病理损伤,牙龈指数、牙槽骨吸收值、MDA、IL-6、TNF-α、Keap1蛋白表达水平明显升高,同时SOD水平、Nrf2蛋白表达水平明显降低,差异有统计学意义(P<0.05);与模型组相比,牙周基础治疗组、片仔癀低剂量+牙周基础治疗组、片仔癀中剂量+牙周基础治疗组、片仔癀高剂量+牙周基础治疗组大鼠牙周组织病理损伤均减轻,牙龈指数、牙槽骨吸收值、MDA、IL-6、TNF-α、Keap1蛋白表达水平均降低,同时SOD水平、Nrf2蛋白表达水平均升高,差异有统计学意义(P<0.05);与牙周基础治疗组相比,片仔癀低剂量+牙周基础治疗组、片仔癀中剂量+牙周基础治疗组、片仔癀高剂量+牙周基础治疗组大鼠牙周组织病理损伤均减轻,牙龈指数、牙槽骨吸收值、MDA、IL-6、TNF-α水平、Keap1蛋白表达水平均降低,同时SOD水平、Nrf2蛋白表达水平均升高,且片仔癀各组呈剂量依赖性,差异有统计学意义(P<0.05)。结论:片仔癀可下调Keap1蛋白表达,上调Nrf2蛋白表达,抑制糖尿病牙周炎大鼠牙周氧化应激及炎症反应,改善其牙槽骨丢失症状。

UPLC-QQQ-MS法同时测定片仔癀中13种成分

目的建立超高效液相色谱串联三重四极杆质谱法(UPLC-QQQ-MS)同时测定片仔癀(牛黄、麝香、三七等)中三七皂苷R1、人参皂苷Re、人参皂苷Rg1、人参皂苷Rb1、人参皂苷Rd、牛磺酸、牛磺胆酸、胆酸、甘氨胆酸、甘氨脱氧胆酸、鹅去氧胆酸、去氧胆酸、麝香酮的含有量。方法该药物甲醇提取液的分析采用Waters CORTECS UPLC C18色谱柱(100 mm×2.1 mm,1.6μm);流动相乙腈-水(含0.1%甲酸),梯度洗脱;柱温40℃;体积流量0.25 m L/min。结果 13种成分在各自范围内线性关系良好(r>0.998 5),平均加样回收率91.1%~105.3%,RSD2.4%~4.6%。结论该方法准确、简便、灵敏、重复性好,可用于片仔癀的质量控制。

片仔癀对人骨肉瘤细胞U2OS细胞周期的影响

目的:探讨片仔癀对体外培养的人骨肉瘤U2OS细胞周期的作用及机制。方法:0.8、1.2、1.6mg/ml片仔癀干预U2OS细胞24、48、72h后,MTT检测片仔癀对U2OS细胞的生长抑制作用;片仔癀干预U2OS细胞48h后流式细胞术分析细胞周期的变化;Western blot检测Survivin蛋白的表达。结果:与对照组比较,不同浓度的片仔癀作用24、48、72h后,不同程度的抑制了U2OS细胞增殖,且呈时间和剂量依赖性(P<0.05);片仔癀干预U2OS细胞48h后细胞发生G2/M阻滞,Survivin蛋白表达较对照组明显降低(P<0.05)。结论:片仔癀可抑制骨肉瘤U2OS细胞的生长,诱导U2OS细胞发生G2/M周期阻滞,周期阻滞的发生与Survivin蛋白表达减少有关。

RP-HPLC测定片仔癀中异嗪皮啶的含量

目的建立复方片仔癀含片中异嗪皮啶HPLC含量测定方法。方法固定相为Nova-pakC18柱,流动相:乙腈-0.1%磷酸溶液(15∶85);流速:1.0mL/min;检测波长:344nm。结果该方法线性范围为12.9～90.3μg(r=0.9999,N=7),平均回收率为100.65%(RSD=0.71%),日内和日间重现性的RSD小于3%。结论该测定方法简便,准确。

片仔癀茵胆平肝胶囊对小鼠镇静催眠的作用

目的:研究片仔癀茵胆平肝脏囊的镇静催眠作用.方法:采用小鼠抖笼滴水法,强迫游泳的不动时间和阈下剂量催眠法.结果:片仔癀茵胆平肝胶囊能显著抑制小鼠的自发活动,延长小鼠强迫游泳的不动时间和增加苯巴比妥阈下剂量的催眠百分率.结论:片仔癀茵胆平肝胶囊具有镇静和对催眠的协同作用.

Pien Tze Huang Inhibits Migration and Invasion of Hepatocellular Carcinoma Cells by Repressing PDGFRB/YAP/CCN2 Axis Activity

Objective:To investigate the effects of Pien Tze Huang(PZH) on the migration and invasion of HCC cells and underlying molecular mechanism.Methods:Cell counting kit-8(CCK-8) was applied to evaluate the cell viabilities of SMMC-7721,SK-Hep-1,C3A and HL-7702(6 × 10^(3)cells/well) co-incubated with different concentrations of PZH(0,0.2,0.4,0.6,0.8 mg/mL) for 24 h.Transwell,wound healing assay,CCK-8and Annexin V-FITC/PI staining were conducted to investigate the effects of PZH on the migration,invasion,proliferation and apoptosis of SK-Hep-1 and SMMC-7721 cells(650 μg/mL for SK-Hep-1 cells and 330 μg/mL for SMMC-7721 cells),respectively.In vivo,lung metastasis mouse model constructed by tail vein injection of HCC cells was used for evaluating the anti-metastasis function of PZH.SK-Hep-1 cells(10^(6)cells/200 μL per mice) were injected into B-NDG mice via tail vein.Totally 8 mice were randomly divided into PZH and control groups,4 mice in each group.After 2-d inoculation,mice in the PZH group were administered with PZH(250 mg/kg,daily) and mice in the control group received only vehicle(PBS) from the 2nd day after xenograft to day 17.Transcriptome analysis based on RNA-seq was subsequently used for deciphering anti-tumor mechanism of PZH.Quantitative real-time polymerase chain reaction(qRT-PCR) and Western blot were applied to verify RNA-seq results.Luciferase reporter assay was performed to examine the transcriptional activity of yes-associated protein(YAP).Results:PZH treatment significantly inhibited the migration,invasion,proliferation and promoted the apoptosis of HCC cells in vitro and in vivo(P<0.01).Transcriptome analysis indicated that Hippo signaling pathway was associated with anti-metastasis function of PZH.Mechanical study showed PZH significantly inhibited the expressions of platelet derived growth factor receptor beta(PDGFRB),YAP,connective tissue growth factor(CCN2),N-cadherin,vimentin and matrix metallopeptidase 2(MMP2,P<0.01).Meanwhile,the phosphorylation of YAP was also enhanced by PZH treatment in vitro and in vivo.Furthermore,PZH played roles in inhibiting the transcriptional activity of YAP.Conclusion:PZH restrained migration,invasion and epithelialmesenchymal transition of HCC cells through repressing PDGFRB/YAP/CCN2 axis.

片仔癀胶囊治疗转移性结肠癌随机双盲对照试验研究

目的评价片仔癀胶囊对转移性结肠癌患者的临床疗效与安全性。方法采用随机双盲安慰剂对照研究方法,将患者按2:1的比例分配至试验组(79例)和对照组(38例),分别给予片仔癀胶囊和安慰剂,每次2粒,每日3次,连续治疗28天为1个疗程,以疾病出现进展为干预终点。检测患者肿瘤标志物(CEA、CA199)、血常规(RBC、WBC、NEU、PLT)及肝肾功能(ALT、AST、γ-GGT、TBIL、BUN、SCr),计算两组无进展生存期(PFS),观察两组患者不良事件(AE)。结果与本组治疗前比较,对照组治疗2个疗程后CA199降低(P<0.01),试验组治疗2个疗程后CEA和CA199降低(P<0.01)。与对照组同期比较,试验组治疗2个疗程后CA199升高(P<0.05)。使用片仔癀胶囊和用药周期≥3个疗程为保护因素(P<0.01,P<0.05)。片仔癀胶囊可提高患者1年无进展生存率(P=0.011)。结论服用片仔癀胶囊3个疗程以上,可降低患者肿瘤标志物CA199,提高PFS,且不增加AE。