菝葜治疗慢性盆腔炎的活性部位筛选及作用机制研究
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摘要
课题背景和目的
慢性盆腔炎疾病(CPID)系指女性内生殖器官、周围结缔组织及盆腔腹膜发生慢性炎症所致。可由急性盆腔炎(PID)治疗不彻底转变而成,这是CPID发病的主要原因；亦可因体质较强,或感邪较轻,受病后表现为症状较轻,病势较缓的慢性盆腔炎。据美国疾病控制和预防中心调查,美国每年150多万妇女患PID,住院费用达10.6亿美元左右,并且每年有上百的PID患者死于并发症。在美国青少年PID感染率比成年妇女高,15～24岁妇女是该疾病高发人群,以美国黑人和拉美裔女性较为常见[2]。据世界卫生组织估计,传播疾病泛滥导致全世界每年新增3亿3千多万性病患者,其中有10%～20%将发展成为CPID。在我国,由于个人卫生条件及医疗条件的限制,或在妇科小手术和计划生育手术中无菌操作不当,加之广泛应用宫内节育器时患者不注意个人卫生等原因,使盆腔炎的发病率逐年提高。同时随着性病在我国的发病率呈逐年升高趋势,由此引起的盆腔炎也在增多。
由于抗生素及手术治疗CPID有许多弊端,中药治疗已日益成为治疗CPID的研究热点。大量文献报道盆益康合剂、黄芪注射液[5]等均能表现出既抗炎、又不易耐药的良好特性,且具有用药安全有效、副作用小、费用较低等优点。金刚藤为菝葜的根部,为传统活血化瘀中药,市售金刚藤胶囊(菝葜提取物)己销售十余年,用于治疗CPID疗效显著,但其制剂较粗、药效机理也未能得很好的阐明。因此,进一步开发菝葜单味药制备成有效部位,并探讨其抗炎及免疫调节作用等与其治疗作用相关的机制具有较高的学术价值和实用临床意义。
本课题通过观察小鼠耳肿胀、大鼠棉球肉芽肿及大鼠慢性盆腔炎疾病(CPID)模型中小鼠耳肿胀度、大鼠棉球肉芽肿抑制率、CPID动物血液学指标及病理改变,对菝葜醇提物及其3个化学部位,即：乙酸乙酯部位、正丁醇部位和水溶液部位的抗炎及抗CPID活性作用进行了比较和筛选,以确定其抗炎和抗CPID的活性部位,并通过柱分离对该部位做进一步的分离纯化,采用HPLC检测其含量对其进行质量评价。在获得了药效确切的活性部位后,亦通过观察其对CPID模型大鼠血清、子宫组织中炎症细胞因子、粘连相关指标的影响,探讨该活性部位对CPID的抗炎、抗粘连的免疫调节机制。本研究力图对菝葜的抗炎及抗CPID的有效物质进行富集,并阐明其药理活性和作用机制,有助于获得药效物质相对明确、药理活性相对特异的菝葜活性部位,为进一步开发菝葜抗CPID有效部位甚至单体化合物制剂提供科学依据。
课题实验方法和结果
1、菝葜的提取分离：采用正交设计L9(34)表,以乙醇浓度、乙醇用量、提取时间为考察因素,以HPLC色谱测定落新妇苷及黄杞苷的含量为考察指标,对菝葜的提取方法进行筛选,并确定菝葜最佳提取工艺。落新妇苷及黄杞苷的含量测定条件为：Hypersil ODS C18色谱柱；流动相：乙腈：0.04%磷酸水；检测波长均为290nm；柱温：室温；流速：0.7m1-min-1。对正交试验所得各工艺样品的落新妇苷和黄杞苷含量分别进行方差分析,结果显示：乙醇浓度对实验结果有显著性影响,其余各因素水平对实验结果的影响均无显著性差异(P>0.05)。随后采用综合平衡法对实验结果进行直观分析,得到菝葜最佳提取工艺为：将菝葜饮片粉碎成小颗粒状,加70%乙醇按乙醇：水为1：8、1：5、1：5提取3次,提取时间分别为2h、2h、1h。提取液过滤后,滤液减压回收乙醇并浓缩至无醇味,置于冰箱中冷藏12h,取出药液,抽滤,合并滤液,加水定容即得,用于后续的研究。
2、菝葜提取液3个化学部位的制备：采用液液萃取方法进行,以乙酸乙酯对提取液萃取得到乙酸乙酯部位,萃取后的药液再以正丁醇萃取得到正丁醇部位,剩下的药液为水溶液部位。乙酸乙酯和正丁醇萃取均采用水饱和的乙酸乙酯和水饱和的正丁醇,按体积比1：1萃取至基本呈无色。采用25%苯酚胶浆0.06ml注入大鼠右侧子宫制备大鼠CPID模型,观察菝葜醇提物及3个化学部位高、中、低(相当于临床等效剂量的4、2、1倍)剂量组对CPID模型大鼠血液学及组织病理形态学改变的影响,探讨其对CPID的治疗作用；采用二甲苯致小鼠耳肿胀法,观察菝葜醇提物及3个化学部位高、中、低(相当于临床等效剂量的4、2、1倍)剂量组对小鼠耳肿胀的抑制作用,探讨其对急性炎症的治疗作用。其中二甲苯用量为每只小鼠右耳涂抹30μl,左耳做空白对照,20min后颈椎脱臼处死小鼠,以直径8mm打孔器分别沿左右耳缘正中相同部位等面积打下两圆形耳片,电子天平称重,计算肿胀度和肿胀抑制率；采用大鼠棉球肉芽肿法,观察菝葜各化学部位高、中、低(相当于临床等效剂量的4、2、1倍)剂量组对大鼠棉球肉芽肿的抑制作用,探讨醇提物及3个化学部位抗慢性炎症作用。其中高压灭菌棉球(每个重50±1mg),临用前用注射器注入头孢美唑1mg/0.1ml,从切口向左侧腹股沟植入皮下。结果显示：菝葜醇提物组与乙酸乙酯部位的高、中剂量组能非常显著地降低CPID模型大鼠增高的血液流变学指标(P<0.01),对小鼠耳肿胀也有非常明显地抑制作用(P<0.01),但低剂量效果均不显著(P>0.05)；乙酸乙酯高剂量组对大鼠棉球肉芽肿也具有非常显著的抑制作用(P<0.01),菝葜醇提物高剂量组和乙酸乙酯部位中、低两个剂量组对大鼠棉球肉芽肿具有显著地抑制作用(P<0.05)。其余各组,包括正丁醇组、水液组的抗炎及抗CPID效果均不明显。镜下观察各组大鼠子宫组织的病理形态学改变,发现：菝葜醇提物组和乙酸乙酯组能明显减少慢性炎症细胞浸润,改善充血、水肿和宫壁各层结构,大部分腺体结构清晰完整,其中中剂量效果不如高剂量明显,低剂量病理改变不明显。正丁醇高剂量组炎症改善不如乙酸乙酯组明显,低剂量组上皮细胞大量脱落至宫腔,炎性细胞覆盖整个黏膜层,散见嗜酸性粒细胞。水液组与模型对照组比较镜下观察无明显差异。上述结果显示：菝葜的乙酸乙酯部位具有较好的抗急、慢性炎症和CPID的作用,故选择该部位进行后续的研究。
3、菝葜活性部位的分离纯化：采用聚酰胺柱对菝葜乙酸乙酯部位进行进一步分离纯化,富集有效成分总黄酮。采用HPLC按“一标多测”法,初步检测其所含有效成分的含量,分析条件为：色谱柱：Hypersil ODS C18(4.6×250nm,51μm)；流动相：甲醇；流速：1ml·min-1；柱温：室温；检测波长：290nm；进样量：20μl。通过“面积归一”法,以落新妇苷的质量(μg)为横坐标,峰面积为纵坐标绘制标准曲线,其回归方程为Y=5.561×105X-5.911×105,R2=0.9997。以峰面积累加,测得乙酸乙酯部位中的总黄酮的含量为55.73%。
4、菝葜活性部位治疗CPID的疗效确证：菝葜按上述方法进行提取、乙酸乙酯萃取,再经聚酰胺树脂纯化后得到了活性部位,其有效成分得到了富集、含量明显提高,其活性强度有可能得到提高,故实验设计时我们降低了大鼠的给药剂量,以临床等效剂量作为中剂量,高、中、低剂量分别相当于临床等效剂量的2、1、1/2倍,较3个化学部位筛选时所用的剂量为低。于CPID造模后第7d开始,每日1次,连续给药10d。于第11d,即末次给药24h后,腹主动脉采血,进行血常规、血流变检测；肉眼观察大鼠子宫造模侧的形态变化；摘取子宫,称重量,计算脏器指数；双侧子宫置10%甲醛固定液浸泡,HE常规染色,镜下观察大鼠子宫的病理改变。结果显示：菝葜活性部位的高、中剂量均能显著降低大鼠血液学指标,包括全血粘度、血浆粘度和白细胞,有非常显著性意义和显著性意义(P<0.01,P<0.01,P<0.05),对大鼠子宫脏器指数的改善也具有统计学意义(P<0.05),但低剂量效果不明显。病理结果显示纯化后的菝葜活性部位低剂量与中药阳性对照组能部分改善CPID模型大鼠子宫的炎症程度,高、中剂量能在很大程度上改善CPID模型大鼠子宫的病变程度,包括对炎症损害的改善作用。上述结果表明：本研究制备得到的菝葜活性部位在临床等效剂量情况下即具有显著地抑制CPID的作用。
5、菝葜活性部位治疗CPID的作用机制研究：通过观察菝葜活性部位对CPID模型大鼠血清、子宫组织中炎症细胞因子、粘连相关指标的变化,探讨其治疗CPID的抗炎抗粘连的免疫调节机制。实验以SD雌性大鼠为研究对象,将84只大鼠随机分为空白对照组、假手术组、模型对照组、菝葜活性部位高、中、低剂量组、金刚藤胶囊组,采用酶联免疫吸附测定法(ELISA)检测各组大鼠血清的炎症细胞因子水平,包括白介素-1p(IL-1β)、白介素-10(IL-10)；免疫组化法检测各组大鼠子宫组织肿瘤坏死因子-α(TNF-α).细胞间粘附分子-1(ICAM-1)的表达；实时荧光定量PCR反应法(RT-PCR)检测大鼠子宫组织TNF-α mRNA及ICAM-1mRNA的表达。结果显示：菝葜活性部位能不同程度地调节模型大鼠血清中IL-1p和IL-10的含量。其中对前炎症细胞因子IL-1β的抑制作用,菝葜活性部位高、中、低3个剂量与模型对照组比较差异具有非常显著性意义(P<0.01)；对抗炎症细胞因子IL-10,菝葜活性部位高、中剂量组能显著升高其在大鼠血清中的含量,差异具有非常显著性意义(P<0.01),低剂量组与模型对照组比较具有显著性差异(P=0.043,<0.05)。通过免疫组化法检测CPID模型大鼠子宫组织中粘连相关指标TNF-α、ICAM-1的表达,结果发现：菝葜活性部位3个剂量组对TNF-α的高表达都有非常显著地抑制作用,而对ICAM-1的抑制作用仅高剂量有非常显著性差异(P<0.01),中、低剂量有显著性差异(P<0.05)。通过RT-PCR法对大鼠子宫TNF-α mRNA及ICAM-1mRNA的表达检测,结果显示：各给药组的TNF-αm RNA及ICAM-1mRNA的表达水平均较模型对照组下降,差异有极显著性意义(P=0.000,<0.001),其中菝葜乙酸乙酯部位高、中剂量组各指标水平均已接近空白对照组水平(P=0.000,<0.001)。本实验的荧光定量PCR扩增结果显示：3个基因扩增曲线光滑平稳,荧光吸收图谱的S形曲线形状完好,符合定量检测要求；融解曲线呈单峰,峰形较锐利,未见其他峰值,提示各基因融解温度均一,扩增产物特异性好,未见非特异的双链DNA产物或引物二聚体。这说明本实验采用RT-PCR所得结果可靠。上述结果表明：本研究制备得到的菝葜活性部位对CPID的抑制作用可能通过抑制前炎症性细胞因子TNF-a和IL-1p,促进抗炎细胞因子IL-10生成,同时抑制粘连相关的粘附分子ICAM-1等的表达与分泌有关。
课题结论
综上所述,本研究通过正交实验优化了菝葜的提取工艺,获得菝葜醇提物及3个化学部位,随后采用CPID大鼠模型、小鼠耳肿胀模型和大鼠棉球肉芽肿模型对上述提取物或化学部位进行了药效学研究,成功筛选出菝葜抗炎及抗CPID活性最强的部位为乙酸乙酯部位,再通过聚酰胺吸附树脂对该化学部位进一步纯化,得到菝葜活性部位,其总黄酮的含量达到55.7%。在CPID大鼠模型上对菝葜活性部位的药理作用做进一步的研究表明：菝葜活性部位可通过降低全血粘度、血浆粘度,缓解CPID大鼠血液高凝和高黏状态,降低大鼠子宫粘连程度,降低毛细血管和细胞膜的通透性,减少炎症渗出及抑制纤维结缔组织增生,促进炎症吸收,从而发挥治疗CPID的作用。机制研究表明,菝葜活性部位抗CPID作用可能与以下调节作用有关：调节大鼠血清炎症细胞因子,即降低促炎因子,升高抗炎因子,从而提高机体抗炎能力,恢复机体免疫平衡；抑制大鼠子宫组织粘连相关指标的表达,减少炎症细胞过度浸润和炎症介质的形成,从而减少黏膜粘连的形成,有效抑制CPID模型大鼠炎症,缓解盆腔粘连。本研究得到的菝葜活性部位,具有药效化学成分相对明确、药理活性相对特异特点,以此为基础,对其作进一步系统的质量分析、药效研究和安全性评价后,可以开发出菝葜抗CPID有效部位现代中药制剂。
Background and objective
The chronic pelvic inflammatory disease (CPID) refers to chronic inflammations of the female internal reproductive organs, surrounding connective tissue and pelvic peritoneum. It may be resulted from the acute pelvic inflammatory disease (PID) treated incompletely; it can also manifest as a mild, moderate chronic pelvic inflammatory disease due to the patient's strong constitution or mild infection. According to the survey of the U.S. Centers for Disease Control and Prevention (CDC), more than150million women in the United States suffered from PID each year, the hospitalization costs amounted to about$1.06billion, and hundreds of PID patients died each year due to complications. In the United States, the infection rate of PID in adolescents is higher than that in adult women,15to24-year-old women are a high-risk population for the diseases, which are more common in African American and Hispanic women. In China, due to personal health and medical conditions, or indifferent aseptic concepts in minor gynecologic surgeries and family planning surgeries, in addition to that IUD are widely used when patients do not pay attention to personal hygiene, the incidence rate of pelvic inflammatory disease is very high. With the increasingly frequent international exchanges, the incidence of sexually transmitted diseases (STD) is increasing year by year in our country, thus it can also lead to an increase in PID.
Since there are many disadvantages in the antibiotics and surgical treatments of CPID, traditional Chinese medicine (TCM) has become a hot spot in the treatment of CPID. A lot of literatures reported that TCM such as Penyikang mixture showed good characteristics of anti-inflammation and not easy to induce drug resistance, and had many advantages including safe and effective medication, small side effect and lower cost. Jingangteng is the root of smilax, a traditional Chinese herb which can promote blood circulation to remove blood stasis, commercially available Jingangteng capsules have been sold for more than10years, and the curative effects are remarkable. Therefore, there are higher academic values in the further exploration of Smilax being prepared into effective fractions and their anti-inflammation and immune regulation mechanism.
In this study, through observing the degree of the mouse ear edema, the inhibition rate of rat cotton ball granuloma, the hematological indices and pathological changes in the models of the mouse ear edema, rat cotton ball granuloma and rat chronic pelvic inflammatory disease (CPID), the activities and roles of anti-inflammation and anti CPID of ethanol extract and three chemical fractions of Smilax china L, which are respectively ethyl acetate fraction, n-butanol fraction and water-soluble fraction, were screened to determine their single active fraction of anti-inflammation and anti-CPID, the fractions were separated and purified by column separation, HPLC were used to detect the contents. And through observing the changes in inflammatory cytokines and adhesions related indicators in the serum and uterine tissues of CPID model rats, the immunomodulatory mechanism of anti-inflammation and anti-adhesion of the active fractions on CPID was explored to make the enrichment degree of effective substance high, the pharmacodynamic material relatively clear, and the pharmacological activity relatively specific, reflecting the scientificity and modernization of traditional Chinese medicine, also providing experimental data for the further development of smilax anti-CPID monomer.
Experimental methods and results
1. Extraction and separation of Smilax:using L9(34) orthogonal design, taking the ethanol concentration, ethanol consumption and extraction time as investigation factors, taking maximum contents of the astilbin and engeletin measured by HPLC as investigation indexes, we screened the methods for ethanol extract and determined the optimal extraction process of Smilax. Hypersil ODS C18column was chosen; mobile phase:acetonitrile:0.04%phosphoric acid; detection wavelengths:290nm; column temperature:room temperature; flow rate:0.7ml·min-1. since the contents of astilbin and engeletin obtained by the orthogonal experiment were estimated by analysis of variance, there were no significant differences in the effects of various factors on the experimental results (P>0.05). Whereupon, the visual analysis of the experimental results were carried out with a comprehensive balance method, the optimal extraction process of Smilax was obtained:Smilax pieces were crushed into small particles, then70%ethanol was added, the extraction was carried out3times by ethanol-water1:8,1:5and1:5, and the extraction times were2h,2h and1h respectively. The filtrate was decompressed to recover the ethanol and concentrated until there was no alcohol flavor, after placed in the refrigerator for12h, it was drained and filtered, and the filtrate was finally collected and added with water at constant volume.
2. Three chemical fractions of Smilax extract were separated by extraction method.
The ethyl acetate fractions were extracted with the extracting solution of ethyl acetate, the drug solution was extracted again with n-butanol to obtain the n-butanol fractions, and the remaining drug solution were water-soluble fractions. Of which, ethyl acetate and n-butanol fractions were extracted7times with water-saturated ethyl acetate and water-saturated n-butanol at a1:1.25%phenol mucilage0.06ml was injected into the right uterus of rats to cause CPID in rats to observe the effects of high, medium and low doses of ethanol extract and three chemical fractions of Smilax china L (corresponding to4,2and1times the clinical equivalent dose respectively) on changes in hematology and histopathology T-phenotype of CPID model rats; xylene was used to induce mouse ear edema to observe the inbibitional effects of high, medium and low doses of Smilax chemical fractions (corresponding to4,2and1times the clinical equivalent dose respectively) on mouse ear edema and explore the therapeutical effects of Smilax chemical fractions on acute inflammations.30μl Xylene was smeared on the right ear of each mouse,whose left ear was used as a blank control,20min later the mice were killed by cervical dislocation, two circular ear pieces were taken with8mm diameter hole puncher at the same middle parts of left and right ear edges and weighed by an electronic balance to calculate the swelling degree and the swelling inhibition rate; rat cotton ball granuloma was induced to observe the inbibitional effects of high, medium and low doses of Smilax chemical fractions (corresponding to4,2and1times the clinical equivalent dose respectively) on rat cotton ball granuloma and explore the anti-chronic inflammatory effects of Smilax chemical fractions. Autoclaved cotton balls (each weighing50±1g) were infused into cefmetazole1mg/0.1ml with a syringe before use, and they were implanted subcutaneously into the rat left groins. Result:The ethanol extract of Smilax and the high, middle doses of the ethyl acetate fractions can significantly reduce the increased hemodynamic indexes of rats, there were significant differences (P<0.01), they could significantly inhibit the mouse ear swelling, there were statistically significant differences (P<0.01), but there were no obvious effects in the low dose group (P>0.05); there was a very significant difference in rat cotton ball granuloma in the high dose group of ethyl acetate (P<0.01), the ethanol extract of Smilax in high dose group and the ethyl acetate fractions in medium, low dose groups had significant differences in rat cotton ball granuloma (P<0.05). The rest groups, including n-butanol fraction and water-soluble fraction groups, had no significant effects of anti-inflammation and anti-CPID. The pathological morphological changes of rat uterine tissues were observed under microscope, the ethanol extract of Smilax and the ethyl acetate fractions could significantly reduce the chronic inflammatory cell infiltration, improve the hyperemia, edema and layer structures of uterine walls, most of the gland structures were clear and complete, among which, the medium dose effects were less obvious than the high dose effects, there were no obvious pathological changes in the low dose group. The inflammation improvement in high dose group of N-butanol was less obvious than that in the ethyl acetate fraction group; in the low-dose group, a large number of epithelial cells fell off into uterine cavity, the inflammatory cells covered the entire mucosal layer, scattered eosinophils were seen. The water-soluble fraction group was compared with the model control group under microscope, and there was no significant difference.
3. The active fractions of Smilax were further separated and purified using polyamide column to collect the effective ingredients of total flavonoids. Using HPLC, chromatographic column:Hypersil ODS C18(4.6×250nm,5μm); mobile phase: methanol; flow rate:1ml·min-1; Column temperature:room temperature; detection wavelength:290nm; sample size:20μl. One single standard substance for determination of multiple constituents (One for M) was used in the initial detection of the active ingredient contents. By area normalization method, the mass of astilbin(μg) as abscissa and the peak area as ordinate, the standard curve was drawn, the regression equation:Y=5.561×105X-5.911×105, R2=0.9997, and the total flavonoids content in ethyl acetate fractions was determined as55.73%by peak area method.
4. Further verification of therapeutic effects of the active fractions of Smilax on CPID: After Smilax active fractions were separated, extracted, extracted with ethyl acetate, and then purified by a polyamide resin, the content was increased, therefore, when designing the experiment, we reduced the administration doses for rats, the clinical equivalent dose was taken as the medium dose, the high, medium and low doses were respectively equal to2,1and1/2times the clinical equivalent dose.7day after rat models were constructed, the drugs were administered once daily for10consecutive days, on the11th day,24h after the last administration, the blood was collected from abdominal aorta to detect the blood count and hemorheological changes; the morphological changes in modeling side of the rat uterus were visually observed; the uterus was taken out and weighed to calculate the viscera index; bilateral uteruses were soaked in10%formaldehyde fixative, then were conventionally stained with HE, the pathological changes of the rat uteruses were observed under microscope. Results:the high and medium doses of active fractions of Smilax can significantly reduce rat hematological indices, including whole blood viscosity, plasma viscosity and white blood cells, there were very significant differences, and a significant difference respectively (P<0.01, P<0.01and P<0.05), the rat uterus viscera index was statistically significant, and the difference was significant (P<0.05), the low dose effect was not obvious. Pathological results showed that the degrees of inflammation of the rat uterus CPID models were partially improved in the low dose group of purified Smilax active fractions and the TCM positive control group, while the pathological damages of the rat uterus CPID models were improved to a great extent in the high and medium dose groups, in addition, the inflammatory damages were improved.
5. The effects of the active fractions of Smilax on inflammatory cytokines and adhesion-related indicators in the serums and uterine tissues of rat CPID models were observed to explore the immunomodulatory mechanism of their anti-inflammatory and anti-adhesion effects on CPID. SD female rats were taken as experimental subjects,84rats were randomly divided into control group, sham operation group, model control group, groups with high, medium and low doses of Smilax active fractions, and Jingangteng capsules group. The enzyme-linked immunosorbent adsorption method (ELISA) was used to detect the inflammatory cytokine levels in the serums of rats in each group, including interleukin-1β (IL-1β) and interleukin-10(IL-10); the immunohistochemical method was used to detect the expression of tumor necrosis factor-a (TNF-a) and intercellular adhesion molecule-1(ICAM-1) in rat uterine tissue of each group; the real time-polymerase chain reaction (RT-PCR) was used to detect the expression of TNF-a mRNA and ICAM-1mRNA in rat uterus tissues. Results:the active fractions of Smilax could regulate the serum IL-1β and IL-10levels of rat models in varying degrees. Of which, there were highly significant differences in regulating the inflammatory cytokines IL-1β between the high, medium and low dose groups of Smilax active fractions and the model control groups (P<0.01); in regulating the inflammatory cytokine IL-10, the active fractions of Smilax in the high and medium dose groups could significantly increase its serum contents in rats, the differences were highly significant (P<0.01), there was a significant difference between the low dose group and the model control group (P=0.043,<0.05). The immunohistochemical method was used to detect the expression of adhesion related indicators such as TNF-a and ICAM-1in the uterine tissues of rat CPID models; the results revealed that the active fractions of Smilax in three dose groups made extremely significant inhibitions on the high expressions of TNF-a, while the active fractions of Smilax made inhibitory effects on the expression of ICAM-1, there was a highly significant difference in high dose group (P<0.01) and significant differences in the medium and low dose groups (P<0.05).The expressions of TNF-a mRNA and of ICAM-1mRNA in rat uterus were detected by RT-PCR method, the results showed that the expressions of TNF-a mRNA and ICAM-1mRNA were decreased in the drug administration groups compared with the model control groups, the differences were highly significant (P=0.000, P<0.001). Of which, the average levels of various indicators in the groups with high and medium doses of ethyl acetate fractions of Smilax had been close to those in the blank control groups (P=0.000,<0.001). At the same time, the experimental results of fluorescence quantitative PCR amplification showed that three gene amplification curves were smooth and stable; the S-shaped curve of the fluorescence absorption spectrum was intact, in line with the requirements of quantitative detection. The melting curve showed a single peak, the peak shape was sharp, and no other peak existed. That indicated that the melting temperature of each gene was homogeneous, the amplification products had good specificities, and no non-specific double-stranded DNA products or primer dimers were seen.
Conclusions
Through studying the pharmacodynamic effects of several chemical fractions of Smilax on CPID rat models, mouse ear edema models and rat cotton ball granuloma models, we found that the active fractions of Smilax with anti-inflammatory and anti-CPID actions were mainly ethyl acetate fractions. Then the active fractions were further separated and purified by polyamide adsorption method, and the total flavonoids content in ethyl acetate fractions was determined as55.73%. The anti-CPID actions of active fractions of Smilax were verified in CPID rat models, this explained that through promoting circulation and removing stasis, the active fractions of Smilax could alleviate the hypercoagulability and hyperviscosity state of blood in CPID rats, reduce the degree of adhesion in the rat uteruses, reduce capillary and cell membrane permeability, reduce inflammatory exudation, inhibit the proliferation of fibrous connective tissues, and promote inflammation absorption. In addition, by regulating inflammatory cytokines in rat serum, the active fractions of Smilax could reduce the pro-inflammatory cytokines and increase the anti-inflammatory cytokines, thereby enhance the body's anti-inflammatory activity to restore the immune balance; by regulating the expression of adhesion-related indicators in rat uteruses,the active fractions of Smilax could reduce the excessive infiltration of inflammatory cells and the formation of inflammatory mediators, reduce the formation of mucosal adhesion, effectively inhibit the inflammations in CPID rat models and alleviate their pelvic adhesions.
慢性盆腔炎疾病(CPID)系指女性内生殖器官、周围结缔组织及盆腔腹膜发生慢性炎症所致。可由急性盆腔炎(PID)治疗不彻底转变而成,这是CPID发病的主要原因；亦可因体质较强,或感邪较轻,受病后表现为症状较轻,病势较缓的慢性盆腔炎。据美国疾病控制和预防中心调查,美国每年150多万妇女患PID,住院费用达10.6亿美元左右,并且每年有上百的PID患者死于并发症。在美国青少年PID感染率比成年妇女高,15～24岁妇女是该疾病高发人群,以美国黑人和拉美裔女性较为常见[2]。据世界卫生组织估计,传播疾病泛滥导致全世界每年新增3亿3千多万性病患者,其中有10%～20%将发展成为CPID。在我国,由于个人卫生条件及医疗条件的限制,或在妇科小手术和计划生育手术中无菌操作不当,加之广泛应用宫内节育器时患者不注意个人卫生等原因,使盆腔炎的发病率逐年提高。同时随着性病在我国的发病率呈逐年升高趋势,由此引起的盆腔炎也在增多。
由于抗生素及手术治疗CPID有许多弊端,中药治疗已日益成为治疗CPID的研究热点。大量文献报道盆益康合剂、黄芪注射液[5]等均能表现出既抗炎、又不易耐药的良好特性,且具有用药安全有效、副作用小、费用较低等优点。金刚藤为菝葜的根部,为传统活血化瘀中药,市售金刚藤胶囊(菝葜提取物)己销售十余年,用于治疗CPID疗效显著,但其制剂较粗、药效机理也未能得很好的阐明。因此,进一步开发菝葜单味药制备成有效部位,并探讨其抗炎及免疫调节作用等与其治疗作用相关的机制具有较高的学术价值和实用临床意义。
本课题通过观察小鼠耳肿胀、大鼠棉球肉芽肿及大鼠慢性盆腔炎疾病(CPID)模型中小鼠耳肿胀度、大鼠棉球肉芽肿抑制率、CPID动物血液学指标及病理改变,对菝葜醇提物及其3个化学部位,即：乙酸乙酯部位、正丁醇部位和水溶液部位的抗炎及抗CPID活性作用进行了比较和筛选,以确定其抗炎和抗CPID的活性部位,并通过柱分离对该部位做进一步的分离纯化,采用HPLC检测其含量对其进行质量评价。在获得了药效确切的活性部位后,亦通过观察其对CPID模型大鼠血清、子宫组织中炎症细胞因子、粘连相关指标的影响,探讨该活性部位对CPID的抗炎、抗粘连的免疫调节机制。本研究力图对菝葜的抗炎及抗CPID的有效物质进行富集,并阐明其药理活性和作用机制,有助于获得药效物质相对明确、药理活性相对特异的菝葜活性部位,为进一步开发菝葜抗CPID有效部位甚至单体化合物制剂提供科学依据。
课题实验方法和结果
1、菝葜的提取分离：采用正交设计L9(34)表,以乙醇浓度、乙醇用量、提取时间为考察因素,以HPLC色谱测定落新妇苷及黄杞苷的含量为考察指标,对菝葜的提取方法进行筛选,并确定菝葜最佳提取工艺。落新妇苷及黄杞苷的含量测定条件为：Hypersil ODS C18色谱柱；流动相：乙腈：0.04%磷酸水；检测波长均为290nm；柱温：室温；流速：0.7m1-min-1。对正交试验所得各工艺样品的落新妇苷和黄杞苷含量分别进行方差分析,结果显示：乙醇浓度对实验结果有显著性影响,其余各因素水平对实验结果的影响均无显著性差异(P>0.05)。随后采用综合平衡法对实验结果进行直观分析,得到菝葜最佳提取工艺为：将菝葜饮片粉碎成小颗粒状,加70%乙醇按乙醇：水为1：8、1：5、1：5提取3次,提取时间分别为2h、2h、1h。提取液过滤后,滤液减压回收乙醇并浓缩至无醇味,置于冰箱中冷藏12h,取出药液,抽滤,合并滤液,加水定容即得,用于后续的研究。
2、菝葜提取液3个化学部位的制备：采用液液萃取方法进行,以乙酸乙酯对提取液萃取得到乙酸乙酯部位,萃取后的药液再以正丁醇萃取得到正丁醇部位,剩下的药液为水溶液部位。乙酸乙酯和正丁醇萃取均采用水饱和的乙酸乙酯和水饱和的正丁醇,按体积比1：1萃取至基本呈无色。采用25%苯酚胶浆0.06ml注入大鼠右侧子宫制备大鼠CPID模型,观察菝葜醇提物及3个化学部位高、中、低(相当于临床等效剂量的4、2、1倍)剂量组对CPID模型大鼠血液学及组织病理形态学改变的影响,探讨其对CPID的治疗作用；采用二甲苯致小鼠耳肿胀法,观察菝葜醇提物及3个化学部位高、中、低(相当于临床等效剂量的4、2、1倍)剂量组对小鼠耳肿胀的抑制作用,探讨其对急性炎症的治疗作用。其中二甲苯用量为每只小鼠右耳涂抹30μl,左耳做空白对照,20min后颈椎脱臼处死小鼠,以直径8mm打孔器分别沿左右耳缘正中相同部位等面积打下两圆形耳片,电子天平称重,计算肿胀度和肿胀抑制率；采用大鼠棉球肉芽肿法,观察菝葜各化学部位高、中、低(相当于临床等效剂量的4、2、1倍)剂量组对大鼠棉球肉芽肿的抑制作用,探讨醇提物及3个化学部位抗慢性炎症作用。其中高压灭菌棉球(每个重50±1mg),临用前用注射器注入头孢美唑1mg/0.1ml,从切口向左侧腹股沟植入皮下。结果显示：菝葜醇提物组与乙酸乙酯部位的高、中剂量组能非常显著地降低CPID模型大鼠增高的血液流变学指标(P<0.01),对小鼠耳肿胀也有非常明显地抑制作用(P<0.01),但低剂量效果均不显著(P>0.05)；乙酸乙酯高剂量组对大鼠棉球肉芽肿也具有非常显著的抑制作用(P<0.01),菝葜醇提物高剂量组和乙酸乙酯部位中、低两个剂量组对大鼠棉球肉芽肿具有显著地抑制作用(P<0.05)。其余各组,包括正丁醇组、水液组的抗炎及抗CPID效果均不明显。镜下观察各组大鼠子宫组织的病理形态学改变,发现：菝葜醇提物组和乙酸乙酯组能明显减少慢性炎症细胞浸润,改善充血、水肿和宫壁各层结构,大部分腺体结构清晰完整,其中中剂量效果不如高剂量明显,低剂量病理改变不明显。正丁醇高剂量组炎症改善不如乙酸乙酯组明显,低剂量组上皮细胞大量脱落至宫腔,炎性细胞覆盖整个黏膜层,散见嗜酸性粒细胞。水液组与模型对照组比较镜下观察无明显差异。上述结果显示：菝葜的乙酸乙酯部位具有较好的抗急、慢性炎症和CPID的作用,故选择该部位进行后续的研究。
3、菝葜活性部位的分离纯化：采用聚酰胺柱对菝葜乙酸乙酯部位进行进一步分离纯化,富集有效成分总黄酮。采用HPLC按“一标多测”法,初步检测其所含有效成分的含量,分析条件为：色谱柱：Hypersil ODS C18(4.6×250nm,51μm)；流动相：甲醇；流速：1ml·min-1；柱温：室温；检测波长：290nm；进样量：20μl。通过“面积归一”法,以落新妇苷的质量(μg)为横坐标,峰面积为纵坐标绘制标准曲线,其回归方程为Y=5.561×105X-5.911×105,R2=0.9997。以峰面积累加,测得乙酸乙酯部位中的总黄酮的含量为55.73%。
4、菝葜活性部位治疗CPID的疗效确证：菝葜按上述方法进行提取、乙酸乙酯萃取,再经聚酰胺树脂纯化后得到了活性部位,其有效成分得到了富集、含量明显提高,其活性强度有可能得到提高,故实验设计时我们降低了大鼠的给药剂量,以临床等效剂量作为中剂量,高、中、低剂量分别相当于临床等效剂量的2、1、1/2倍,较3个化学部位筛选时所用的剂量为低。于CPID造模后第7d开始,每日1次,连续给药10d。于第11d,即末次给药24h后,腹主动脉采血,进行血常规、血流变检测；肉眼观察大鼠子宫造模侧的形态变化；摘取子宫,称重量,计算脏器指数；双侧子宫置10%甲醛固定液浸泡,HE常规染色,镜下观察大鼠子宫的病理改变。结果显示：菝葜活性部位的高、中剂量均能显著降低大鼠血液学指标,包括全血粘度、血浆粘度和白细胞,有非常显著性意义和显著性意义(P<0.01,P<0.01,P<0.05),对大鼠子宫脏器指数的改善也具有统计学意义(P<0.05),但低剂量效果不明显。病理结果显示纯化后的菝葜活性部位低剂量与中药阳性对照组能部分改善CPID模型大鼠子宫的炎症程度,高、中剂量能在很大程度上改善CPID模型大鼠子宫的病变程度,包括对炎症损害的改善作用。上述结果表明：本研究制备得到的菝葜活性部位在临床等效剂量情况下即具有显著地抑制CPID的作用。
5、菝葜活性部位治疗CPID的作用机制研究：通过观察菝葜活性部位对CPID模型大鼠血清、子宫组织中炎症细胞因子、粘连相关指标的变化,探讨其治疗CPID的抗炎抗粘连的免疫调节机制。实验以SD雌性大鼠为研究对象,将84只大鼠随机分为空白对照组、假手术组、模型对照组、菝葜活性部位高、中、低剂量组、金刚藤胶囊组,采用酶联免疫吸附测定法(ELISA)检测各组大鼠血清的炎症细胞因子水平,包括白介素-1p(IL-1β)、白介素-10(IL-10)；免疫组化法检测各组大鼠子宫组织肿瘤坏死因子-α(TNF-α).细胞间粘附分子-1(ICAM-1)的表达；实时荧光定量PCR反应法(RT-PCR)检测大鼠子宫组织TNF-α mRNA及ICAM-1mRNA的表达。结果显示：菝葜活性部位能不同程度地调节模型大鼠血清中IL-1p和IL-10的含量。其中对前炎症细胞因子IL-1β的抑制作用,菝葜活性部位高、中、低3个剂量与模型对照组比较差异具有非常显著性意义(P<0.01)；对抗炎症细胞因子IL-10,菝葜活性部位高、中剂量组能显著升高其在大鼠血清中的含量,差异具有非常显著性意义(P<0.01),低剂量组与模型对照组比较具有显著性差异(P=0.043,<0.05)。通过免疫组化法检测CPID模型大鼠子宫组织中粘连相关指标TNF-α、ICAM-1的表达,结果发现：菝葜活性部位3个剂量组对TNF-α的高表达都有非常显著地抑制作用,而对ICAM-1的抑制作用仅高剂量有非常显著性差异(P<0.01),中、低剂量有显著性差异(P<0.05)。通过RT-PCR法对大鼠子宫TNF-α mRNA及ICAM-1mRNA的表达检测,结果显示：各给药组的TNF-αm RNA及ICAM-1mRNA的表达水平均较模型对照组下降,差异有极显著性意义(P=0.000,<0.001),其中菝葜乙酸乙酯部位高、中剂量组各指标水平均已接近空白对照组水平(P=0.000,<0.001)。本实验的荧光定量PCR扩增结果显示：3个基因扩增曲线光滑平稳,荧光吸收图谱的S形曲线形状完好,符合定量检测要求；融解曲线呈单峰,峰形较锐利,未见其他峰值,提示各基因融解温度均一,扩增产物特异性好,未见非特异的双链DNA产物或引物二聚体。这说明本实验采用RT-PCR所得结果可靠。上述结果表明：本研究制备得到的菝葜活性部位对CPID的抑制作用可能通过抑制前炎症性细胞因子TNF-a和IL-1p,促进抗炎细胞因子IL-10生成,同时抑制粘连相关的粘附分子ICAM-1等的表达与分泌有关。
课题结论
综上所述,本研究通过正交实验优化了菝葜的提取工艺,获得菝葜醇提物及3个化学部位,随后采用CPID大鼠模型、小鼠耳肿胀模型和大鼠棉球肉芽肿模型对上述提取物或化学部位进行了药效学研究,成功筛选出菝葜抗炎及抗CPID活性最强的部位为乙酸乙酯部位,再通过聚酰胺吸附树脂对该化学部位进一步纯化,得到菝葜活性部位,其总黄酮的含量达到55.7%。在CPID大鼠模型上对菝葜活性部位的药理作用做进一步的研究表明：菝葜活性部位可通过降低全血粘度、血浆粘度,缓解CPID大鼠血液高凝和高黏状态,降低大鼠子宫粘连程度,降低毛细血管和细胞膜的通透性,减少炎症渗出及抑制纤维结缔组织增生,促进炎症吸收,从而发挥治疗CPID的作用。机制研究表明,菝葜活性部位抗CPID作用可能与以下调节作用有关：调节大鼠血清炎症细胞因子,即降低促炎因子,升高抗炎因子,从而提高机体抗炎能力,恢复机体免疫平衡；抑制大鼠子宫组织粘连相关指标的表达,减少炎症细胞过度浸润和炎症介质的形成,从而减少黏膜粘连的形成,有效抑制CPID模型大鼠炎症,缓解盆腔粘连。本研究得到的菝葜活性部位,具有药效化学成分相对明确、药理活性相对特异特点,以此为基础,对其作进一步系统的质量分析、药效研究和安全性评价后,可以开发出菝葜抗CPID有效部位现代中药制剂。
Background and objective
The chronic pelvic inflammatory disease (CPID) refers to chronic inflammations of the female internal reproductive organs, surrounding connective tissue and pelvic peritoneum. It may be resulted from the acute pelvic inflammatory disease (PID) treated incompletely; it can also manifest as a mild, moderate chronic pelvic inflammatory disease due to the patient's strong constitution or mild infection. According to the survey of the U.S. Centers for Disease Control and Prevention (CDC), more than150million women in the United States suffered from PID each year, the hospitalization costs amounted to about$1.06billion, and hundreds of PID patients died each year due to complications. In the United States, the infection rate of PID in adolescents is higher than that in adult women,15to24-year-old women are a high-risk population for the diseases, which are more common in African American and Hispanic women. In China, due to personal health and medical conditions, or indifferent aseptic concepts in minor gynecologic surgeries and family planning surgeries, in addition to that IUD are widely used when patients do not pay attention to personal hygiene, the incidence rate of pelvic inflammatory disease is very high. With the increasingly frequent international exchanges, the incidence of sexually transmitted diseases (STD) is increasing year by year in our country, thus it can also lead to an increase in PID.
Since there are many disadvantages in the antibiotics and surgical treatments of CPID, traditional Chinese medicine (TCM) has become a hot spot in the treatment of CPID. A lot of literatures reported that TCM such as Penyikang mixture showed good characteristics of anti-inflammation and not easy to induce drug resistance, and had many advantages including safe and effective medication, small side effect and lower cost. Jingangteng is the root of smilax, a traditional Chinese herb which can promote blood circulation to remove blood stasis, commercially available Jingangteng capsules have been sold for more than10years, and the curative effects are remarkable. Therefore, there are higher academic values in the further exploration of Smilax being prepared into effective fractions and their anti-inflammation and immune regulation mechanism.
In this study, through observing the degree of the mouse ear edema, the inhibition rate of rat cotton ball granuloma, the hematological indices and pathological changes in the models of the mouse ear edema, rat cotton ball granuloma and rat chronic pelvic inflammatory disease (CPID), the activities and roles of anti-inflammation and anti CPID of ethanol extract and three chemical fractions of Smilax china L, which are respectively ethyl acetate fraction, n-butanol fraction and water-soluble fraction, were screened to determine their single active fraction of anti-inflammation and anti-CPID, the fractions were separated and purified by column separation, HPLC were used to detect the contents. And through observing the changes in inflammatory cytokines and adhesions related indicators in the serum and uterine tissues of CPID model rats, the immunomodulatory mechanism of anti-inflammation and anti-adhesion of the active fractions on CPID was explored to make the enrichment degree of effective substance high, the pharmacodynamic material relatively clear, and the pharmacological activity relatively specific, reflecting the scientificity and modernization of traditional Chinese medicine, also providing experimental data for the further development of smilax anti-CPID monomer.
Experimental methods and results
1. Extraction and separation of Smilax:using L9(34) orthogonal design, taking the ethanol concentration, ethanol consumption and extraction time as investigation factors, taking maximum contents of the astilbin and engeletin measured by HPLC as investigation indexes, we screened the methods for ethanol extract and determined the optimal extraction process of Smilax. Hypersil ODS C18column was chosen; mobile phase:acetonitrile:0.04%phosphoric acid; detection wavelengths:290nm; column temperature:room temperature; flow rate:0.7ml·min-1. since the contents of astilbin and engeletin obtained by the orthogonal experiment were estimated by analysis of variance, there were no significant differences in the effects of various factors on the experimental results (P>0.05). Whereupon, the visual analysis of the experimental results were carried out with a comprehensive balance method, the optimal extraction process of Smilax was obtained:Smilax pieces were crushed into small particles, then70%ethanol was added, the extraction was carried out3times by ethanol-water1:8,1:5and1:5, and the extraction times were2h,2h and1h respectively. The filtrate was decompressed to recover the ethanol and concentrated until there was no alcohol flavor, after placed in the refrigerator for12h, it was drained and filtered, and the filtrate was finally collected and added with water at constant volume.
2. Three chemical fractions of Smilax extract were separated by extraction method.
The ethyl acetate fractions were extracted with the extracting solution of ethyl acetate, the drug solution was extracted again with n-butanol to obtain the n-butanol fractions, and the remaining drug solution were water-soluble fractions. Of which, ethyl acetate and n-butanol fractions were extracted7times with water-saturated ethyl acetate and water-saturated n-butanol at a1:1.25%phenol mucilage0.06ml was injected into the right uterus of rats to cause CPID in rats to observe the effects of high, medium and low doses of ethanol extract and three chemical fractions of Smilax china L (corresponding to4,2and1times the clinical equivalent dose respectively) on changes in hematology and histopathology T-phenotype of CPID model rats; xylene was used to induce mouse ear edema to observe the inbibitional effects of high, medium and low doses of Smilax chemical fractions (corresponding to4,2and1times the clinical equivalent dose respectively) on mouse ear edema and explore the therapeutical effects of Smilax chemical fractions on acute inflammations.30μl Xylene was smeared on the right ear of each mouse,whose left ear was used as a blank control,20min later the mice were killed by cervical dislocation, two circular ear pieces were taken with8mm diameter hole puncher at the same middle parts of left and right ear edges and weighed by an electronic balance to calculate the swelling degree and the swelling inhibition rate; rat cotton ball granuloma was induced to observe the inbibitional effects of high, medium and low doses of Smilax chemical fractions (corresponding to4,2and1times the clinical equivalent dose respectively) on rat cotton ball granuloma and explore the anti-chronic inflammatory effects of Smilax chemical fractions. Autoclaved cotton balls (each weighing50±1g) were infused into cefmetazole1mg/0.1ml with a syringe before use, and they were implanted subcutaneously into the rat left groins. Result:The ethanol extract of Smilax and the high, middle doses of the ethyl acetate fractions can significantly reduce the increased hemodynamic indexes of rats, there were significant differences (P<0.01), they could significantly inhibit the mouse ear swelling, there were statistically significant differences (P<0.01), but there were no obvious effects in the low dose group (P>0.05); there was a very significant difference in rat cotton ball granuloma in the high dose group of ethyl acetate (P<0.01), the ethanol extract of Smilax in high dose group and the ethyl acetate fractions in medium, low dose groups had significant differences in rat cotton ball granuloma (P<0.05). The rest groups, including n-butanol fraction and water-soluble fraction groups, had no significant effects of anti-inflammation and anti-CPID. The pathological morphological changes of rat uterine tissues were observed under microscope, the ethanol extract of Smilax and the ethyl acetate fractions could significantly reduce the chronic inflammatory cell infiltration, improve the hyperemia, edema and layer structures of uterine walls, most of the gland structures were clear and complete, among which, the medium dose effects were less obvious than the high dose effects, there were no obvious pathological changes in the low dose group. The inflammation improvement in high dose group of N-butanol was less obvious than that in the ethyl acetate fraction group; in the low-dose group, a large number of epithelial cells fell off into uterine cavity, the inflammatory cells covered the entire mucosal layer, scattered eosinophils were seen. The water-soluble fraction group was compared with the model control group under microscope, and there was no significant difference.
3. The active fractions of Smilax were further separated and purified using polyamide column to collect the effective ingredients of total flavonoids. Using HPLC, chromatographic column:Hypersil ODS C18(4.6×250nm,5μm); mobile phase: methanol; flow rate:1ml·min-1; Column temperature:room temperature; detection wavelength:290nm; sample size:20μl. One single standard substance for determination of multiple constituents (One for M) was used in the initial detection of the active ingredient contents. By area normalization method, the mass of astilbin(μg) as abscissa and the peak area as ordinate, the standard curve was drawn, the regression equation:Y=5.561×105X-5.911×105, R2=0.9997, and the total flavonoids content in ethyl acetate fractions was determined as55.73%by peak area method.
4. Further verification of therapeutic effects of the active fractions of Smilax on CPID: After Smilax active fractions were separated, extracted, extracted with ethyl acetate, and then purified by a polyamide resin, the content was increased, therefore, when designing the experiment, we reduced the administration doses for rats, the clinical equivalent dose was taken as the medium dose, the high, medium and low doses were respectively equal to2,1and1/2times the clinical equivalent dose.7day after rat models were constructed, the drugs were administered once daily for10consecutive days, on the11th day,24h after the last administration, the blood was collected from abdominal aorta to detect the blood count and hemorheological changes; the morphological changes in modeling side of the rat uterus were visually observed; the uterus was taken out and weighed to calculate the viscera index; bilateral uteruses were soaked in10%formaldehyde fixative, then were conventionally stained with HE, the pathological changes of the rat uteruses were observed under microscope. Results:the high and medium doses of active fractions of Smilax can significantly reduce rat hematological indices, including whole blood viscosity, plasma viscosity and white blood cells, there were very significant differences, and a significant difference respectively (P<0.01, P<0.01and P<0.05), the rat uterus viscera index was statistically significant, and the difference was significant (P<0.05), the low dose effect was not obvious. Pathological results showed that the degrees of inflammation of the rat uterus CPID models were partially improved in the low dose group of purified Smilax active fractions and the TCM positive control group, while the pathological damages of the rat uterus CPID models were improved to a great extent in the high and medium dose groups, in addition, the inflammatory damages were improved.
5. The effects of the active fractions of Smilax on inflammatory cytokines and adhesion-related indicators in the serums and uterine tissues of rat CPID models were observed to explore the immunomodulatory mechanism of their anti-inflammatory and anti-adhesion effects on CPID. SD female rats were taken as experimental subjects,84rats were randomly divided into control group, sham operation group, model control group, groups with high, medium and low doses of Smilax active fractions, and Jingangteng capsules group. The enzyme-linked immunosorbent adsorption method (ELISA) was used to detect the inflammatory cytokine levels in the serums of rats in each group, including interleukin-1β (IL-1β) and interleukin-10(IL-10); the immunohistochemical method was used to detect the expression of tumor necrosis factor-a (TNF-a) and intercellular adhesion molecule-1(ICAM-1) in rat uterine tissue of each group; the real time-polymerase chain reaction (RT-PCR) was used to detect the expression of TNF-a mRNA and ICAM-1mRNA in rat uterus tissues. Results:the active fractions of Smilax could regulate the serum IL-1β and IL-10levels of rat models in varying degrees. Of which, there were highly significant differences in regulating the inflammatory cytokines IL-1β between the high, medium and low dose groups of Smilax active fractions and the model control groups (P<0.01); in regulating the inflammatory cytokine IL-10, the active fractions of Smilax in the high and medium dose groups could significantly increase its serum contents in rats, the differences were highly significant (P<0.01), there was a significant difference between the low dose group and the model control group (P=0.043,<0.05). The immunohistochemical method was used to detect the expression of adhesion related indicators such as TNF-a and ICAM-1in the uterine tissues of rat CPID models; the results revealed that the active fractions of Smilax in three dose groups made extremely significant inhibitions on the high expressions of TNF-a, while the active fractions of Smilax made inhibitory effects on the expression of ICAM-1, there was a highly significant difference in high dose group (P<0.01) and significant differences in the medium and low dose groups (P<0.05).The expressions of TNF-a mRNA and of ICAM-1mRNA in rat uterus were detected by RT-PCR method, the results showed that the expressions of TNF-a mRNA and ICAM-1mRNA were decreased in the drug administration groups compared with the model control groups, the differences were highly significant (P=0.000, P<0.001). Of which, the average levels of various indicators in the groups with high and medium doses of ethyl acetate fractions of Smilax had been close to those in the blank control groups (P=0.000,<0.001). At the same time, the experimental results of fluorescence quantitative PCR amplification showed that three gene amplification curves were smooth and stable; the S-shaped curve of the fluorescence absorption spectrum was intact, in line with the requirements of quantitative detection. The melting curve showed a single peak, the peak shape was sharp, and no other peak existed. That indicated that the melting temperature of each gene was homogeneous, the amplification products had good specificities, and no non-specific double-stranded DNA products or primer dimers were seen.
Conclusions
Through studying the pharmacodynamic effects of several chemical fractions of Smilax on CPID rat models, mouse ear edema models and rat cotton ball granuloma models, we found that the active fractions of Smilax with anti-inflammatory and anti-CPID actions were mainly ethyl acetate fractions. Then the active fractions were further separated and purified by polyamide adsorption method, and the total flavonoids content in ethyl acetate fractions was determined as55.73%. The anti-CPID actions of active fractions of Smilax were verified in CPID rat models, this explained that through promoting circulation and removing stasis, the active fractions of Smilax could alleviate the hypercoagulability and hyperviscosity state of blood in CPID rats, reduce the degree of adhesion in the rat uteruses, reduce capillary and cell membrane permeability, reduce inflammatory exudation, inhibit the proliferation of fibrous connective tissues, and promote inflammation absorption. In addition, by regulating inflammatory cytokines in rat serum, the active fractions of Smilax could reduce the pro-inflammatory cytokines and increase the anti-inflammatory cytokines, thereby enhance the body's anti-inflammatory activity to restore the immune balance; by regulating the expression of adhesion-related indicators in rat uteruses,the active fractions of Smilax could reduce the excessive infiltration of inflammatory cells and the formation of inflammatory mediators, reduce the formation of mucosal adhesion, effectively inhibit the inflammations in CPID rat models and alleviate their pelvic adhesions.
引文
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