中药熏洗对膝骨性关节炎患者关节滑液中MMP-3和MMP-13的影响

目的:观察中药熏洗治疗膝关节骨性关节炎的临床疗效和对关节滑液中MMP-3和MMP-13的影响。方法:膝关节骨性关节炎90例,分成治疗组和对照组。分别采用补肾活血汤熏洗和热开水熏洗加双氯芬酸钠乳膏外搽治疗,观察两组患者的临床症状、体征、Lysholm's膝关节评分以及关节滑液中MMP-3和MMP-13变化。结果:两组治疗后Lysholm's膝关节评分较治疗前明显升高(P<0.01),治疗组的评分值高于对照组(P<0.01),治疗组总体优良率为93.33%,对照组总体优良率为82.22%,治疗组疗效优于对照组(P<0.01),两组治疗后关节滑液MMP-3、MMP-13水平明显下降(P<0.01),治疗组MMP-3、MMP-13下降较对照组更为明显(P<0.01)。结论:补肾活血汤熏洗治疗膝骨性关节炎疗效显著,其作用机制可能通过降低MMP-3、MMP-13的水平,减轻滑膜炎症和关节软骨的破坏,从而发挥对骨性关节炎的治疗作用。

Expression of MMP-2 and MMP-13 in Bullous Pemphigoid

Objective:To investigate the expression and significance of matrix metalloproteinase-2(MMP-2)and matrix metalloproteinase-13(MMP-13)in bullous pemphigoid(BP)skin lesions.Methods:Immunohistochemical SP method was used to detect the expression of MMP-2 and MMP-13 in 32 BP skin lesions,and compared with 15 normal skin tissues.Results:The expression of MMP-2 in the case group was significantly increased(38.56±10.06)compared to the normal control group(21.20±5.98);the expression of MMP-13 in the case group was significantly augmented(18.62±5.90)compared to the normal control group(11.47±8.484).The expressions of MMP-2 and MMP-13 in the skin lesions of patients with bullous pemphigoid were statistically different from those of normal people(both P<0.05).Compared with the expression of MMP-2 and MMP-13 in bullous pemphigoid,the expression of MMP-2 and MMP-13 was moderately correlated(correlation coefficient was 0.523).Conclusion:The expression of MMP-2 and MMP-13 is significantly increased in bullous pemphigoid skin lesions,suggesting that they may play an important role in the pathogenesis of BP.There is a certain correlation between the expression of MMP-2 and MMP-13,suggesting that the high expression of MMP-13 may play a role in the mechanism that further leads to the high expression of MMP-2.

牙龈卟啉单胞菌感染对牙龈上皮细胞中基质金属蛋白酶13表达的影响

目的通过体外建立牙龈卟啉单胞菌(P.gingivalis)感染牙龈上皮细胞模型,探讨P.gingivalis W83、ATCC 33277感染牙龈上皮细胞后基质金属蛋白酶13(MMP-13)表达的变化。方法将P.gingivalis W83、ATCC 33277作用于牙龈上皮细胞6、12、24和48 h,采用ELISA检测上清液中MMP-13蛋白浓度变化,实时PCR检测MMP-13 mRNA相对含量变化。结果 P.gingivalis感染牙龈上皮细胞后,MMP-13蛋白和mRNA表达的总体趋势是随时间的延长表达水平逐渐增高。P.gingivalis感染牙龈上皮细胞6 h,P.gingivalis ATCC 33277感染组和W83感染组MMP-13蛋白表达水平与对照组相比无统计学差异,而MMP-13 mRNA的相对表达量高于对照组(P<0.05);感染24和48 h后,P.gingivalis ATCC 33277感染组和W83感染组MMP-13蛋白和mRNA的相对表达量均明显高于对照组(P<0.05),且P.gingivalis W83感染组MMP-13明显高于P.gingivalis ATCC 33277感染组(P<0.05)。结论 P.gingivalis能够促进牙龈上皮细胞分泌MMP-13,造成牙周组织破坏。P.gingivalis W83降解细胞外基质的能力高于P.gingivalis ATCC 33277,更利于细菌向深部组织扩散。

Structure-based Screening for the Non-zinc-chelating Selective MMP-13 Inhibitors of Natural Products

Matrix metalloproteinase-13(MMP-13) has been considered as a promising therapeutic target for osteoarthritis. In this work, the experimental crystal structures of five MMP-13-ligand complexes are used to build the multiple structure-based pharmacophore model of MMP-13 inhibitors. The reliability of pharmacophore model is validated using a decoy set. The pharmacophore model contains four chemical features: two hydrogen bond acceptor(HBA), one hydrophobic(HY) feature, and one ring aromatic(RA) feature. Particularly, the HY feature is found to orient the MMP-13 inhibitors deep into the S1’ pocket of MMP-13 to produce selective inhibition. By carrying out the screening of pharmacophore model and subsequent molecular docking, the four non-zinc-chelating selective MMP-13 inhibitors of natural products(NP-015973, NP-000814, STOCK1 N-24933, and STOCK1 N-69443) are identified. It is found that the binding modes of MMP-13 with our screened four natural products are very similar to the reported experimental binding mode of MMP-13 with the most active inhibitor(GG12003, IC50: 0.67 n M), and each of them involves the interactions of a ligand with the three amino acid residues Thr226, Lys119, and His201 of MMP-13 receptor. This shows that our modeling results are in good agreement with the relevant experimental results, which strongly supports our screened MMP-13 inhibitors of natural products. These screened natural products may be used as the lead compounds of MMP-13 inhibitors in the future studies of structural modifications.

纳米金光热作用对ICR小鼠皮肤光老化保护作用的初步研究

目的探讨纳米金光热作用对雌性ICR小鼠皮肤光老化的保护作用及其可能机制。方法32只健康雌性ICR小鼠随机分成正常组、模型组、红光组、纳米金组，每组8只。正常组小鼠不予任何干预，另3组每次照射前背部脱毛，面积约16cm^2。每次照射紫外线前，模型组暂不予以特殊处理。红光组小鼠给予红光照射，纳米金组小鼠先涂抹纳米金球溶液，用黑色塑料薄膜封包30min后再照射红光。经上述处理后，将模型组、红光组、纳米金组小鼠分笼放入灯箱中进行紫外线照射。实验结束后，肉眼及皮肤镜观察皮肤外观变化；取小鼠背部皮肤，组织切片HE染色和Masson染色观察皮肤组织结构和胶原纤维的改变。ImageJ图像分析软件计算胶原纤维面积。DCFH—DA探针检测皮肤细胞内活性氧簇（ROS）含量。ELISA检测皮肤组织中基质金属蛋白酶13（MMP-13）的含量。采用单因素方差分析进行组间差异分析，并用LSD—t检验进行两两多重比较。用简单线性回归分析ROS变化与MMP-13表达量之间的相关性。结果与正常组比较，紫外线照射后，模型组小鼠皮肤出现皮屑、褐黄、粗糙肥厚、深皱纹、缺乏弹性等光老化特征；皮肤镜下小鼠皮肤粗糙，毛细血管断裂出血，结痂，褐色斑点。组织切片显示，模型组小鼠表皮厚度[（81．32±7．09）μm]和真皮厚度[（352．75±40．08）μm]均较正常组[表皮（38．42±13．64）μm，真皮（188．50±27．83）μm]明显增加（P〈0．05），胶原纤维面积减少（分别为31．94％±8．56％和48．63％±11．32％，P〈0．05），异常弹性物质增多；模型组ROS含量（5124152．80±754119．22）DCF荧光强度／mg蛋白和MMP-13表达（3895．42±136．06）g／L均较正常组（分别为3502085．29±135089．06）DCF荧光强度／mg蛋白和（2414．73±105．96）g/L明显增高（P〈0．05）。与模型组比较，纳米金组皮肤外观及组织学特点均明显改善，小鼠皮肤厚度明显变薄，表皮（48．70±13．35）μm，真皮（253．74±36．28）μm（P〈0．05），且其表皮厚度恢复正常（P〉0．05），胶原纤维面积（41．54％±12．10％）增加（P〈0．05）；纳米金组ROS含量（3516963．35±67436．36）DCF荧光强度／mg蛋白和MMP-13表达（2558．25±102．72）g/L均较模型组明显降低（P〈0．05），且恢复至正常水平。用简单线性回归分析显示，ROS的产生量与MMP-13的表达量呈正相关（r=0．864，P=0．018）。结论纳米金的光热作用对小鼠光老化有一定的保护作用，其机制可能与其减少ICR小鼠皮肤组织中ROS产生，从而减少下游MMP-13表达有关。