阿魏酸钠抗炎分子机制及对奶牛子宫内膜炎疗效初步观察
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摘要
子宫内膜炎（Endometritis）是奶牛常见疾病，是奶牛不孕不育的主要原因。奶牛患子宫内膜炎导致人工授精次数增加，配种和受孕间隔延长，妊娠率、受孕率降低等，使奶牛淘汰率提高，造成养牛业巨大的损失。经流行病学调查，发现东北地区奶牛临床子宫内膜炎的平均发病率为17.4%。主要的致病菌为大肠杆菌。兽医临床上治疗子宫内膜炎多数使用抗生素注射或药物子宫内灌注，这些方法虽能获得较好的治疗效果，但会在乳品中形成药残，出现抗药性，从而危及人体健康。中药有提高动物机体免疫机能、抵抗疾病的能力，同时中药具有毒性低、无残留、不易产生耐药性的优点，克服了抗生素类药的缺点。因此人们在积极探寻治疗子宫内膜炎的中药，防止“有抗奶”的发生。
经过对大量国内、外资料的查阅，我们发现中药当归、川芎中最重要的有效成份之一阿魏酸钠具有抗炎作用，但阿魏酸钠是否具有抗奶牛子宫内膜炎的作用，其抗炎分子机制又如何？为了进一步阐明这些问题，我们对其进行了深入研究，探讨其抗炎机理以及观察其治疗子宫内膜炎的临床效果，为进一步研发抗炎中药方剂和配伍提供理论和基础实验数据。
本论文首先建立LPS诱导的小鼠单核-巨噬细胞RAW264.7的体外炎症模型，进行了不同浓度条件下阿魏酸钠对RAW264.7细胞的细胞因子TNF-α，IL-1β和IL-6及炎性介质NO和PGE2分泌的影响。实验表明，阿魏酸钠能够调控TNF-α、IL-6、IL-1β的分泌，并呈现剂量依赖性。另外，阿魏酸钠还能调节NO、PGE2的合成。从以上结果可知，在动物体外研究中，阿魏酸钠的抗炎作用是通过调控细胞因子和炎性介质的分泌而进行的。我们又对LPS诱导细胞因子及炎性介质产生的机制进行了研究，采用LPS刺激小鼠的RAW264.7细胞，测定阿魏酸钠对第二信使[Ca~(2+)]i、cAMP/cGMP信号通路、NF-κB和MAPKs信号转导通路的影响。由实验可知，阿魏酸钠抑制了LPS刺激的小鼠RAW264.7巨噬细胞[Ca~(2+)]i浓度、NF-κB的活力和ERK、JNK、p38MAPKs蛋白表达，其作用呈剂量依赖方式，使cAMP浓度升高，但对cGMP无影响。结果表明，阿魏酸钠通过调控[Ca~（~(2+)）]i、cAMP、NF-κB、ERK、JNK和p38MAPKs通路，抑制细胞因子TNF-α、IL-6、IL-1β分泌和炎性介质NO、PGE_2合成。
在体外实验的基础上，我们又进行阿魏酸钠体内抗炎实验。在LPS诱导的大鼠子宫内膜炎模型中，我们研究了阿魏酸钠的治疗作用。结果显示，阿魏酸钠能明显减轻大鼠子宫内膜的炎症反应，治疗组子宫指数与正常对照组差异不显著，子宫组织显微结构显示基本正常，表明阿魏酸钠对实验性子宫内膜炎模型大鼠有较好的治疗效果。
最后，我们进行了治疗奶牛子宫内膜炎临床实验，结果表明，阿魏酸钠治疗临床型子宫内膜炎后受孕率达83.3％，而土霉素治疗的受孕率为66.7％，在受孕率上阿魏酸钠的治疗效果好于土霉素。在隐性子宫内膜炎治疗上，阿魏酸钠组受孕率达62.5％，而土霉素组受孕率为12.5％，阿魏酸钠与土霉素相比差异显著(P＜0.05)。
Endometritis of dairy cow is a common disease in dairy cows, also is one of themain causes of infertility. The number of artificial insemination, mating andpregnancy prolongation,the cow culling rate were increased by suffering fromendometritis.,meanwhile,pregnancy rate was decreased.All of them to be reduced cancause a huge losses in the cattle industry.we found that the average clinicalendometritis disease incidence is17.4%in the epidemiological investigation inNortheast China. The main pathogens is staphylococcus. The majority of endometritistreatment on veterinary clinic use antibiotic injections or medicine intrauterineinfusion. Although these methods can obtain good therapeutic effect,it will be causeddrug residues in milk, the emergence of drug-resistant, thereby endangering the healthof human bodies. Traditional chinese medicine can improve immune function, animalresistance to disease, and there is no resistance, drug residues and toxic side effects ofantibiotics.So people is actively exploring traditional Chinese medicine in thetreatment of endometritis, eliminating body resistance.
Through investigation,we found that sodium ferulate,one most important of theactive ingredients of the Chinese angelica, has anti-inflammatory effects.But whethersodium ferulate can resist the effect of cow endometritis,how about theanti-inflammatory molecular mechanism is? So we explored the anti-inflammatorymechanisms deeply and clinical effect of the treatment of endometritis, It is providedtheoretical foundation and the basis of experimental data for further research anddevelopment of anti-inflammatory medicine prescription and compatibility.
Firstly, we studied the effect of different concentrations of sodium ferulate oncytokines TNF-α, IL-1β, IL-6on RAW264.7cells. The result showed that sodiumferulate inhibite TNF-α, IL-6and IL-1β secretion level in a dose-dependent manner.In addition, sodium ferulate also inhibited NO and PGE2synthesis in LPS-stimulatedmurine RAW264.7macrophages by inhibiting iNOS and COX-2protein expression,cytosolic free Ca~(2+)([Ca~(2+)]i), and increasing cAMP level.The above data show that, in vitro, sodium ferulate can exert anti-inflammatory effect by the regulation of secretionof cytokines and inflammatory mediat.NF-κB and MAPKs are known as importanttargets for anti-inflammatory molecular mechanism.In order to studyanti-infalmmatory molecular mechanism, we further investigated the effect of sodiumferulate on NF-κB and MAPKs signal transductive pathways in LPS-stimulatedmurine RAW264.7macrophages.The result showed that sodium ferulate inhibitedNF-κB activity, ERK、JNK and p38protein phosphorylation in a dose-dependentmanner. It suggested that sodium ferulate inhibited cytokine TNF-α, IL-1β, IL-6, NOand PGE2secretion by regulating both NF-κB and ERK and p38MAPKs pathways.
Secondly,in vitro experiments, we further validate the anti-inflammatory effectsof ferulic sodium in the body. LPS-induced rat uterine meningitis model, we study thetherapeutic effect of sodium ferulate.The results show that sodium ferulate cansignificantly reduce the inflammatory response in rat endometrium, uterus index ofthe treatment group and the normal control group had no significant difference,basically normal uterine tissue microstructure,it shows that ferulic sodiumexperimental temper intrauterine the meningitis model rats have a better therapeuticeffect.
Finally,we conducted a clinical experimental treatment of endometritis,resultsshowed that conception rate was83.3%in treatment of sodium ferulate,whilepregnancy of oxytetracycline treatment was66.7%.The pregnancy rate of ferulicsodium treatment better than oxytetracycline. In the treatment of chronic endometritis,conception rate was62.5%in sodium ferulate group, while conception rate was12.5%in the oxytetracycline group, The result showed significant difference in sodiumferulate and oxytetracycline (P＜0.05).
经过对大量国内、外资料的查阅，我们发现中药当归、川芎中最重要的有效成份之一阿魏酸钠具有抗炎作用，但阿魏酸钠是否具有抗奶牛子宫内膜炎的作用，其抗炎分子机制又如何？为了进一步阐明这些问题，我们对其进行了深入研究，探讨其抗炎机理以及观察其治疗子宫内膜炎的临床效果，为进一步研发抗炎中药方剂和配伍提供理论和基础实验数据。
本论文首先建立LPS诱导的小鼠单核-巨噬细胞RAW264.7的体外炎症模型，进行了不同浓度条件下阿魏酸钠对RAW264.7细胞的细胞因子TNF-α，IL-1β和IL-6及炎性介质NO和PGE2分泌的影响。实验表明，阿魏酸钠能够调控TNF-α、IL-6、IL-1β的分泌，并呈现剂量依赖性。另外，阿魏酸钠还能调节NO、PGE2的合成。从以上结果可知，在动物体外研究中，阿魏酸钠的抗炎作用是通过调控细胞因子和炎性介质的分泌而进行的。我们又对LPS诱导细胞因子及炎性介质产生的机制进行了研究，采用LPS刺激小鼠的RAW264.7细胞，测定阿魏酸钠对第二信使[Ca~(2+)]i、cAMP/cGMP信号通路、NF-κB和MAPKs信号转导通路的影响。由实验可知，阿魏酸钠抑制了LPS刺激的小鼠RAW264.7巨噬细胞[Ca~(2+)]i浓度、NF-κB的活力和ERK、JNK、p38MAPKs蛋白表达，其作用呈剂量依赖方式，使cAMP浓度升高，但对cGMP无影响。结果表明，阿魏酸钠通过调控[Ca~（~(2+)）]i、cAMP、NF-κB、ERK、JNK和p38MAPKs通路，抑制细胞因子TNF-α、IL-6、IL-1β分泌和炎性介质NO、PGE_2合成。
在体外实验的基础上，我们又进行阿魏酸钠体内抗炎实验。在LPS诱导的大鼠子宫内膜炎模型中，我们研究了阿魏酸钠的治疗作用。结果显示，阿魏酸钠能明显减轻大鼠子宫内膜的炎症反应，治疗组子宫指数与正常对照组差异不显著，子宫组织显微结构显示基本正常，表明阿魏酸钠对实验性子宫内膜炎模型大鼠有较好的治疗效果。
最后，我们进行了治疗奶牛子宫内膜炎临床实验，结果表明，阿魏酸钠治疗临床型子宫内膜炎后受孕率达83.3％，而土霉素治疗的受孕率为66.7％，在受孕率上阿魏酸钠的治疗效果好于土霉素。在隐性子宫内膜炎治疗上，阿魏酸钠组受孕率达62.5％，而土霉素组受孕率为12.5％，阿魏酸钠与土霉素相比差异显著(P＜0.05)。
Endometritis of dairy cow is a common disease in dairy cows, also is one of themain causes of infertility. The number of artificial insemination, mating andpregnancy prolongation,the cow culling rate were increased by suffering fromendometritis.,meanwhile,pregnancy rate was decreased.All of them to be reduced cancause a huge losses in the cattle industry.we found that the average clinicalendometritis disease incidence is17.4%in the epidemiological investigation inNortheast China. The main pathogens is staphylococcus. The majority of endometritistreatment on veterinary clinic use antibiotic injections or medicine intrauterineinfusion. Although these methods can obtain good therapeutic effect,it will be causeddrug residues in milk, the emergence of drug-resistant, thereby endangering the healthof human bodies. Traditional chinese medicine can improve immune function, animalresistance to disease, and there is no resistance, drug residues and toxic side effects ofantibiotics.So people is actively exploring traditional Chinese medicine in thetreatment of endometritis, eliminating body resistance.
Through investigation,we found that sodium ferulate,one most important of theactive ingredients of the Chinese angelica, has anti-inflammatory effects.But whethersodium ferulate can resist the effect of cow endometritis,how about theanti-inflammatory molecular mechanism is? So we explored the anti-inflammatorymechanisms deeply and clinical effect of the treatment of endometritis, It is providedtheoretical foundation and the basis of experimental data for further research anddevelopment of anti-inflammatory medicine prescription and compatibility.
Firstly, we studied the effect of different concentrations of sodium ferulate oncytokines TNF-α, IL-1β, IL-6on RAW264.7cells. The result showed that sodiumferulate inhibite TNF-α, IL-6and IL-1β secretion level in a dose-dependent manner.In addition, sodium ferulate also inhibited NO and PGE2synthesis in LPS-stimulatedmurine RAW264.7macrophages by inhibiting iNOS and COX-2protein expression,cytosolic free Ca~(2+)([Ca~(2+)]i), and increasing cAMP level.The above data show that, in vitro, sodium ferulate can exert anti-inflammatory effect by the regulation of secretionof cytokines and inflammatory mediat.NF-κB and MAPKs are known as importanttargets for anti-inflammatory molecular mechanism.In order to studyanti-infalmmatory molecular mechanism, we further investigated the effect of sodiumferulate on NF-κB and MAPKs signal transductive pathways in LPS-stimulatedmurine RAW264.7macrophages.The result showed that sodium ferulate inhibitedNF-κB activity, ERK、JNK and p38protein phosphorylation in a dose-dependentmanner. It suggested that sodium ferulate inhibited cytokine TNF-α, IL-1β, IL-6, NOand PGE2secretion by regulating both NF-κB and ERK and p38MAPKs pathways.
Secondly,in vitro experiments, we further validate the anti-inflammatory effectsof ferulic sodium in the body. LPS-induced rat uterine meningitis model, we study thetherapeutic effect of sodium ferulate.The results show that sodium ferulate cansignificantly reduce the inflammatory response in rat endometrium, uterus index ofthe treatment group and the normal control group had no significant difference,basically normal uterine tissue microstructure,it shows that ferulic sodiumexperimental temper intrauterine the meningitis model rats have a better therapeuticeffect.
Finally,we conducted a clinical experimental treatment of endometritis,resultsshowed that conception rate was83.3%in treatment of sodium ferulate,whilepregnancy of oxytetracycline treatment was66.7%.The pregnancy rate of ferulicsodium treatment better than oxytetracycline. In the treatment of chronic endometritis,conception rate was62.5%in sodium ferulate group, while conception rate was12.5%in the oxytetracycline group, The result showed significant difference in sodiumferulate and oxytetracycline (P＜0.05).
引文
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