乳酸抑制精液来源淀粉样纤维的形成

摘要

Objective To investigate the inhibitory effect of lactic acid on semen-derived amyloid (SEVI) fibril formation. Methods PAP248-286 (2 mg/mL) was incubated with 4.0, 2.0, 1.0, 0.5, 0.25, and 0.125 mg/mL of lactic acid. After incubation for different times, aliquots were drawn from each sample for Thioflavin T (ThT) and Congo red staining to monitor semen-derived amyloid fibril formation. The β sheet structure formation of PAP248-286 was measured by circular dichroism spectrum, and the morphology of amyloid fibrils incubated with or without lactic acid was observed with transmission electron microscopy (TEM). The enhancing effect of amyloid fibril incubated with lactic acid at different time points was determined using virus infection assay. PAP248-286 (2 mg/mL) was incubated with dilutions of vaginal secretion from healthy women, and amyloid fibril formation was detected with ThT and Congo red staining. Results Lactic acid inhibited SEVI fibril formation in a dose-dependent manner in vitro. Lactic acid at 0.5 mg/mL completely inhibited 2 mg/mL SEVI fibril formation within 48 h. After incubation for 48 h, lactic acid at 1 mg/mL inhibited the formation ofβ-sheet structure of SEVI (2 mg/mL) and completely inhibited 2 mg/mL PAP248-286 aggregation as observed with TEM. In the presence of lactic acid, PAP248-286 lost the ability to enhance virus infection. Vaginal secretion inhibited SEVI fibril formation in a dose-dependent manner, and virtually no SEVI fibril occurred after incubation of 2 mg/mL PAP248-286 with 67%vaginal secretion. Conclusion Lactic acid inhibits SEVI fibril formation in vitro.%目的 探究乳酸对精液来源的淀粉样纤维形成的抑制作用.方法 2 mg/mL的PAP248-286多肽与不同浓度乳酸(4、2、1、0.5、0.25、0.125 mg/mL)分别孵育,在不同时间点取样,用硫磺素T和刚果红染色法分别检测淀粉样纤维形成的过程;采用圆二色谱法检测乳酸对PAP248-286形成β-片层结构的影响;采用透射电子显微镜观察乳酸对淀粉样纤维形态的影响;用病毒感染实验,检测与乳酸孵育的不同时间点,精液淀粉样纤维促进病毒感染的能力;将2 mg/mL的PAP248-286多肽与不同浓度的健康女性阴道分泌液孵育后,用硫磺素T和刚果红染色法评价阴道分泌液抑制精液淀粉样纤维形成的作用.结果 乳酸体外能浓度依赖性地抑制精液淀粉样纤维的形成,在48 h内,0.5 mg/mL乳酸能够抑制2 mg/mL的淀粉样纤维SEVI的形成;在48 h内,1 mg/mL的乳酸能抑制精液淀粉样纤维(2 mg/mL)的β-片层结构的形成,透射电子显微镜观察到1 mg/mL的乳酸能完全抑制2 mg/mL PAP248-286聚合形成淀粉样纤维;与乳酸孵育的多肽PAP248-286,失去了促进病毒感染的能力;阴道分泌液浓度依赖性地抑制SEVI的形成,48 h内,2 mg/mL的PAP248-286在67%阴道分泌液存在时几乎无SEVI生成.结论 乳酸能抑制精液来源的淀粉样纤维的形成.