Luo H, Huang N, Yang M, Tang B, Wu Q, Wang B
College of Stomatology, West China University of Medical Sciences.
Hua Xi Kou Qiang Yi Xue Za Zhi. 2000 Oct;18(5):301-3.
Human parotid histidine-rich-polypeptides (HRPs) are a family of lowmolecular-weight, cationic polypeptides. HRP-1, HRP-3 and HRP-5 comprise 85%-90% of the total HRPs and are called major HRPs. There were many researches indicating the antimicrobial activities of HRPs. Recently, HRPs were reported to have an inhibitory action on the lipopolysaccharide (LPS) of E. coli, with HRP-5 being the most effective one among the three major HRPs. Since the LPS of oral gram-negative bacteria is thought to be one of the important etiological factors during the development of periodontal diseases, our experiment was aimed to investigate the neutralizing effect of human parotid HRP-5 on the LPS of anaerobic suspected periodontal pathogens, which have different chemical structures and biological activities compared with LPS of aerobic E. coli.
By using of preparative acid urea polyacrylamide gel electrophoresis (AU-PAGE), HRP-5 was purified from parotid saliva collected from healthy adults. Two stains of suspected periodontal pathogens, Porphyromonas gingivalis (P.g, 47-A) and Fusobacterium nuceatum (F. n, separated from subgingival plaque of a patient with adult periodontitis) were mass cultured. After harvested in the midlogarithmic phase, they were washed and lyophilized. The LPS of dried bacteria were extracted by the modified Westphal hot phenol-water procedures and purified by the enzyme digestion plus ultracentrifugation. Limulus test was applied to test the neutralizing effect of HRP-5 on the LPS-induced gelation of Limulus amoebocyte lysate. In brief, the standard LPS of E. coli, or extracted LPS of P. g or F. n, was preincubated with HRP-5 in a tube separately in room temperature for 10 minutes. Then the reagent of Limulus amoebocyte lysate was added in all the tubes, continued the incubation in 37 degrees C for one hour. After that, the gelation level of every tube was observed.
LPS extracted from P. g and F. n both showed good purity and strong activities to induce gelation of Limulus amoebocyte lysate. The gelation induced by LPS (1 ng/ml) of these two anaerobic suspected periodontal pathogens were weakly inhibited by HRP-5 (10 micrograms/ml), similar to that observed with standard LPS of E. coli. To get a complete neutralizing effect on LPS, it may be important to increase the concentration of HRP-5.
It appeared that HRPs could neutralize the endotoxic properties of LPS of suspected periodontal pathogens, therefore may contribute to periodontal health. The present investigation further confirmed that HRPs are important components of the host non-immune defense system.
人腮腺富含组氨酸的多肽（HRPs）是一类低分子量的阳离子多肽家族。HRP-1、HRP-3和HRP-5占总HRPs的85%-90%，被称为主要HRPs。有许多研究表明HRPs具有抗菌活性。最近，有报道称HRPs对大肠杆菌的脂多糖（LPS）有抑制作用，其中HRP-5是三种主要HRPs中最有效的一种。由于口腔革兰氏阴性菌的LPS被认为是牙周疾病发生过程中的重要致病因素之一，我们的实验旨在研究人腮腺HRP-5对厌氧疑似牙周病原体LPS的中和作用，这些病原体的化学结构和生物学活性与需氧大肠杆菌的LPS不同。
采用制备性酸性尿素聚丙烯酰胺凝胶电泳（AU-PAGE）从健康成年人腮腺唾液中纯化HRP-5。对两种疑似牙周病原体菌株，牙龈卟啉单胞菌（P.g，47-A）和具核梭杆菌（F.n，从一名成人牙周炎患者的龈下菌斑中分离）进行大量培养。在对数中期收获后，将它们洗涤并冻干。通过改良的Westphal热酚-水法提取干燥细菌的LPS，并通过酶消化加超速离心进行纯化。采用鲎试剂法检测HRP-5对LPS诱导的鲎血细胞溶解物凝胶化的中和作用。简而言之，将大肠杆菌的标准LPS或提取的P.g或F.n的LPS分别与HRP-5在室温下于试管中预孵育10分钟。然后在所有试管中加入鲎血细胞溶解物试剂，在37℃继续孵育1小时。之后，观察每个试管的凝胶化程度。
从P.g和F.n中提取的LPS均显示出良好的纯度和较强的诱导鲎血细胞溶解物凝胶化的活性。这两种厌氧疑似牙周病原体的LPS（1 ng/ml）诱导的凝胶化受到HRP-5（10微克/ml）的微弱抑制，类似于对大肠杆菌标准LPS的观察结果。要对LPS产生完全的中和作用，增加HRP-5的浓度可能很重要。
似乎HRPs可以中和疑似牙周病原体LPS的内毒素特性，因此可能有助于牙周健康。本研究进一步证实HRPs是宿主非免疫防御系统的重要组成部分。
