四氢嘧啶类化合物ZL-5015的免疫抑制作用及其作用机制研究
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摘要
目的
新型四氢嘧啶类化合物ZL-5015(1,3-二环戊基-1,2,3,6-四氢嘧啶-4,5-、甲酸二乙酯)兼具抗炎和免疫抑制活性,本研究采用体内和体外实验模型探讨ZL-5015的免疫抑制作用特点及作用机制,为后续的结构改造及深入的作用机制研究提供参考信息。
研究内容
1.ZL-5015对正常小鼠免疫功能的影响
1.1对体液免疫功能的影响---兔红细胞免疫小鼠产生血清溶血素(IgM)模型
1.1.1材料与方法
BALB/c小鼠50只,随机分为5组,各组动物均连续7天给药,给药后第2天上午,每只鼠腹腔注射20%(V/V)的兔红细胞生理盐水0.2ml进行免疫,同日下午继续给药,免疫后第6天(免疫当天为第0天),动物摘眼球取血,分光光度法检测各组小鼠血清中的溶血素水平。
1.1.2结果
ZL-5015高、中剂量组(200.100mg/kg.b.w)能明显抑制小鼠血清溶血素水平,抑制率分别为32.8%和20.0%(P=0.000,P=0.000),ZL-5015低剂量组(50mg/kg.b.w)能轻微抑制小鼠血清溶血素水平,但与溶媒对照组比较差异无统计学意义(P=0.131)。
1.2对细胞免疫功能的影响---二硝基氟苯(DNFB)诱导小鼠迟发型超敏反应(DTH)模型
1.2.1材料与方法
BALB/c小鼠50只,随机分为5组,给药第一天用1%DNFB致敏小鼠,连续给药7天,于致敏后第7天,取1%DNFB丙酮-橄榄油(1：1)20gL均匀涂布于每只小鼠右耳的两面进行攻击。于攻击24h后,给小鼠称重,颈椎脱臼处死,剪下左右耳廓,以左右耳片质量差值作为肿胀度,并计算肿胀率。取出脾脏和胸腺称重并计算相应的器官指数。
1.2.2结果
ZL-5015高、中、低剂量组(200、100、50mg/kg.b.w)与模型组比较,均能显著抑制二硝基氟苯诱导的小鼠耳廓肿胀(P=0.000,P=0.000,P=0.015),抑制率分别为：56.45%、47.58%、29.84%。ZL-5015高剂量(200mg/kg.b.w)能抑制小鼠的脾脏指数(P=0.013),中、低剂量组(100、50mg/kg.b.w)无抑制作用(P=0.482,P=0.418)。ZL-5015高、中、低剂量组(200、100.50mg/kg.b.w)对小鼠的胸腺指数无显著影响(P=0.384,P=0.527,P=0.570)。
1.3ZL-5015对网状内皮细胞(巨噬细胞)吞噬功能的影响(碳粒廓清实验)
1.3.1材料与方法
BALB/c小鼠50只,随机分为5组,连续给药7天。末次给药1h后,先称小鼠体重,每只小鼠尾静脉均注射印度墨汁(用生理盐水稀释6倍),按0.1ml/10g注射。分别于注射后于1min(t1)和10min(t10)小鼠眼球取血,检测OD450值,最后将小鼠颈椎脱臼处死,同时摘取小鼠肝脏、脾脏,并称湿重,计算廓清指数(K)及吞噬指数(A)。
1.3.2结果
化合物ZL-5015高、中、低剂量组(200、100、50mg/kg.b.w)对小鼠吞噬指数A无明显影响(P=1.000,P=0.999,P=1.000)。
2.ZL-5015对大鼠佐剂性关节炎模型的影响
2.1ZL-5015对大鼠佐剂性关节炎模型关节炎指数、足趾肿胀度、体重的影响
2.1.1材料与方法
雌性SD大鼠48只,随机分为6组,每只鼠左后足趾皮内注射100μL弗氏佐剂,致炎后第16天开始给药和测量,共给药12天。采用排水法测足部体积：在大鼠足部踝骨关节突出部位划一条线作为标记,用足趾肿胀仪测量,间隔4天测一次足趾肿胀情况及体重。SD大鼠免疫后,根据大鼠四肢关节炎症的红、肿及硬化程度,计算多发性关节炎指数。
2.1.2结果
致炎后第20天,ZL-5015高剂量(200mg/kg.b.w)处理组关节炎指数与模型组比较明显下降,下降率为20.0%(P=0.010),说明此时高剂量药物开始起效。对足趾肿胀及体重无显著影响。
致炎后第24天,ZL-5015高、中、低剂量(200、100、50mg/kg.b.w)使关节炎指数分别下降34.5%、25.0%、19.0%(P=0.000,P=0.001,P=0.010)。高剂量能使足趾肿胀度下降49.5%(P=0.048),中、低剂量使足趾肿胀度分别下降40.4%及21.5%,但无统计学意义(P=0.194,P=0.845)。对体重有增重趋势,但差异无统计学意义。
致炎后第28天,ZL-5015高、中、低剂量(200、100、50mg/kg.b.w)使关节炎指数分别下降43.0%、36.7%、26.6(P=0.000,P=0.000,P=0.001)。高、中剂量使足趾肿胀分别下降54.0%及48.7%(P=0.034,P=0.041),低剂量下降39.4%,但差异无统计学意义(P=0.150)。对体重有增重趋势,但差异无统计学意义。
2.2ZL-5015对弗氏佐剂关节炎大鼠血清促炎细胞因子的影响
2.2.1材料与方法
于佐剂关节炎造模第28天,股动脉取血,3000rpm离心10min,取上清,ELISA方法检测IL-1β及TNF-α活性
2.2.2结果
ZL-5015高、中剂量(200、100mg/kg)与模型组比较,均可显著降低关节炎大鼠血清IL-1β的水平(P=0.006,P=0.049),且有浓度依赖趋势,抑制率分别为：27.0%,19.1%；ZL-5015低剂量组(50mg/kg)可以轻微降低大鼠血清IL-1β的水平(P=0.426)。ZL-5015高、中剂量组(200、100mg/kg)均可以显著降低大鼠血清TNF-a的水平(P=0.000,P=0.005),且有浓度依赖趋势,抑制率分别为：29.2%,21.6%；ZL-5015低剂量组(50mg/kg可以轻微降低大鼠血清TNF-α的水平,但差异无统计学意义(P=0.173)。
2.3ZL-5015对关节炎部位促炎细胞因子的影响
2.3.1材料与方法
于造模第28天,大鼠断头处死,在非致炎侧踝关节上方0.5cm处摘取肿胀的足爪,剪碎,生理盐水浸泡过夜,离心取上清,ELISA方法检测IL-1β及TNF-a活性。
2.3.2结果
ZL-5015高、中剂量(200、100mg/kg)与模型组比较,均可显著减少关节炎部位IL-1β的含量(P=0.000,P=0.019),且有浓度依赖趋势,抑制率分别为：30.3%,19.3%；ZL-5015低剂量(50mg/kg)可以轻微减少炎症部位IL-1β的含量,但差异无统计学意义(P=0.123)。ZL-5015高、中、低剂量组(200、100、50mg/kg)均可显著减少炎症部位TNF-α的含量(P=0.000,P=0.004,P=0.039),且有浓度依赖趋势,抑制率分别为：31.9%,22.0%和15.2%。
3.ZL-5015对淋巴细胞功能的影响
3.1ZL-5015对Con A和LPS诱导小鼠脾细胞增殖反应的影响
3.1.1材料与方法
Balb/c小鼠两只,常规制备脾细胞悬液,Con A(4μg/ml)和LPS(10μg/ml)刺激小鼠脾细胞增殖作为体外免疫模型,检测给药48小时后各组细胞的代谢活力。
3.1.2结果
ZL-5015高、中、低剂量均可以显著抑制Con A诱导的小鼠脾细胞增殖反应,(P=0.000,P=0.000,P=0.000),且有浓度依赖趋势,抑制率分别是：33.10%、21.0%和13.28%。ZL-5015高、中剂量组均可以显著抑制LPS诱导的小鼠脾细胞增殖反应(P=0.000,P=0.000),且有浓度依赖趋势,抑制率分别是：25.90%、14.95%,ZL-5015低剂量组有轻微抑制LPS诱导的小鼠脾细胞增殖反应的趋势,但差异无统计学意义(P=0.087)。
3.2ZL-5015对混合淋巴细胞培养反应的影响
3.2.1材料与方法
将BALB/c小鼠和C57BL/6J小鼠的脾细胞分别制成悬液,按照1：1混合制成细胞浓度为6x106个/mL的细胞悬液,按分组加入药物,用96孔培养板培养72h后各组细胞的代谢活力。
3.2.2结果
ZL-5015高、中、低剂量组均可以显著抑制混合淋巴细胞的增殖反应(P=0.000,P=0.000,P=0.037),且有浓度依赖趋势,抑制率分别是：37.2%、17.4%和5.0%。
3.3ZL-5015对Con A刺激小鼠脾细胞分泌白介素2和干扰素γ的影响
3.3.1材料与方法
Balb/c小鼠两只,常规制备脾细胞悬液,Con A(4μg/ml)刺激小鼠脾细胞活化,检测给药48小时后各组细胞中上清液白细胞介素2和干扰素γ的含量。
3.3.2结果
ZL-5015高、中、低剂量组(40、20、10μM)均可显著抑制Con A诱导的小鼠脾细胞分泌白介素2(P=0.000,P=0.000,P=0.007),且有浓度依赖趋势,抑制率分别是：29.37%、20.31%和11.35%。ZL-5015高、中、低剂量组(40、20、10μM)均可以显著抑制Con A诱导的小鼠脾细胞分泌干扰素γ(P=0.000,P=0.002,P=0.030),且有浓度依赖趋势,抑制率分别是：31.9%、23.6%和15.9%。
3.4ZL-5015对小鼠脾细胞细胞因子mRNA表达的抑制作用
3.4.1材料与方法
Balb/c小鼠两只,常规制备脾细胞悬液,Con A(4μg/ml)刺激小鼠脾细胞活化,使用荧光定量PCR检测给药48小时后各组细胞中白细胞介素2和干扰素γmRNA表达的变化。
3.4.2结果
ZL-5015(40.20.10μM)可抑制由Con A诱导的IL-2mRNA的表达(P=0.010,P=0.024,P=0.048)。ZL-5015(40、20、10μM)可抑制由Con A诱导的INF-γmRNA的表达(P=0.005,P=0.013,P=0.043)。
3.5ZL-5015对LPS刺激小鼠脾细胞分泌IgG1和IgG2a的影响
3.5.1材料与方法
Balb/c小鼠两只,常规制备脾细胞悬液,LPS(10μg/ml)激小鼠脾细胞活化,各组给药作用48小时后,收集培养上清液检测IgGl和IgG2a的含量。
3.5.2结果
ZL-5015高、中剂量组(40、20μM)均可以显著抑制LPS诱导的小鼠脾细胞分泌IgGl,(P=0.000,P=0.001),且有浓度依赖趋势,抑制率分别是：27.8%、18.0%；ZL-5015低剂量组(10μM)可以轻微抑制LPS诱导的小鼠脾细胞分泌IgGl,但差异无统计学意义(P=0.105)。ZL-5015高、中、低剂量组(40、20、10μM)均可以显著抑制LPS诱导的小鼠脾细胞分泌IgG2a(P=0.000,P=0.000,P=0.011),且有浓度依赖趋势,抑制率分别是：31.6%、22.7%和13.7%。
3.6ZL-5015对T细胞亚类的影响
3.6.1材料与方法
脾细胞制备、实验分组及给药均同3.5.1所述,48小时后收集细胞,用荧光标记的抗小鼠CD3、CD4、 CD8抗体染色,流式细胞仪检测。统计方法
3.6.2结果
ZL-5015主要降低Con A诱导的CD4弱表达和CD3弱表达T细胞亚群(CD4LowCD3Low)的比例。
统计学处理
应用SPSS11.5统计软件进行统计分析。T细胞亚类占细胞总数的比例以百分数表示,先采用RxC表资料的卡方检验对各组细胞的百分率差异进行多组间的总体检验,然后采用四格表资料的卡方检验进行组间细胞百分率的两两比较。定量数据结果以均数±标准差表示,采用单向方差分析法分析,先进行方差齐性检验,若方差齐用LSD方法多重比较；若方差不齐,采用Welch F检验和Dunnett's T3法多重比较。显著性水准取α=0.05,以P<0.05时,组间差异具有统计学意义。
结论
1、ZL-5015可降低兔红细胞诱导小鼠产生血清溶血素的活性,降低二硝基氟苯诱导的小鼠耳廓的肿胀度,对小鼠碳粒廓清的速率无明显影响,提示对小鼠的体液免疫及细胞免疫功能有抑制作用,对非特异性免疫功能无影响。
2、ZL-5015可以减轻弗氏佐剂关节炎模型的关节炎指数,足趾肿胀,提示对类风湿关节炎具有一定的临床应用前景。
3、ZL-5015可以减少弗氏佐剂关节炎模型血清及炎症部位中的促炎细胞因子IL-1β和TNF-α的含量,提示对佐剂性关节炎的治疗作用与抑制促炎细胞因子的产生与关。
4、ZL-5015可抑制Con A和LPS诱导的小鼠脾细胞的增殖反应,提示对T淋巴细胞和B淋巴细胞的增殖反应有直接的抑制作用。
5、ZL-5015可以显著抑制混合淋巴细胞的增殖反应(代谢活力),提示对特异抗原诱导的淋巴细胞增殖反应有直接的抑制作用。
6、ZL-5015可以显著抑制Con A诱导的小鼠脾细胞分泌白介素2和干扰素γ,提示其免疫抑制作用与抑制细胞因子的产生与分泌有关。
7、ZL-5015可抑制由Con A诱导的IL-2及INF-y mRNA的表达,提示其抑制细胞因子产生的作用环节在mRNA表达水平。
8、ZL-5015可以显著抑制LPS诱导的小鼠脾细胞分泌IgGl和IgG2a,提示其可直接作用于B淋巴细胞而抑制抗体的产生。
9、ZL-5015可降低Con A诱导的CD4弱表达和CD3弱表达T细胞亚群(CD4LowCD3Low)的比例,提示其作用机制可能是抑制CD4+CD3+细胞的活化与增殖,从而抑制免疫反应。
10、总之,ZL-5015对体液和细胞免疫反应均有抑制作用,对T和B淋巴细胞的增殖与分泌功能具有直接抑制作用,对自身免疫性疾病动物模型有效,但需要高剂量(100mg/kg以上),与现有临床药物相比其作用强度还不够理想,需要进一步结构改造。
Objective
The novel tetrahydropyrimidine compound ZL-5015(1,3-dicyclopentyl-1,2,3,6-tetrahydropyrimidine-4,5-dicarboxylic acid diethyl ester) possesses both anti-inflammatory and immune suppressive activities. This study was undertaken to investigate its immunosuppressive characteristics and the underlying mechanisms in vivo and in vitro in an attempt to provide relevant information for further structure modification and research.
Research content
1. Effect of ZL-5015on mouse immune function
1.1Effect of ZL-5015on humoral immune function---Evaluated by the level of serum hemolysin (IgM) in mice immunized with rabbit red blood cells.
1.1.1Materials and Methods
Fifty male and female mice were equitably and randomly assigned into5groups (n=10/group). The mice were intragastrically administered with ZL-5015for7consecutive days. On the second day, the animals were primed with0.2mL rabbit red blood cells (RRBC,20%v/v, per mouse, i.p.). After the treatment on the last day, blood was collected from the fossa orbitalis plexus venosus of the animals and the level of hemolysin was determined by spectrophotometry.
1.1.2Results
ZL-5015(200,100and50mg/kg) reduced the hemolysin level in sera in mice induced by RRBC in a dose dependent manner with the inhibition rates of32.8%(P＜0.001),20.0%(P＜0.001), and8.1%(P＞0.05), respectively.
1.2Effect of ZL-5015on cellular immune function---Evaluated by delayed type hypersensitivity (DTH) induced by DNFB
1.2.1Materials and Methods
Fifty male and female mice were equitably and randomly assigned into5groups (n=10/group).On the first day after intragastric administration of ZL-5015, the mice were primed with DNFB by applying20μL1%DNFB in1:1acetone-olive oil to the hair removed area of the animal abdomen (2cm×2cm). ZL-5015was administered once a day for7consecutive days. On the sixth day,20μL1%DNFB solution was used to challenge the left ear of the mice by applying evenly to the dorsal and ventral surfaces of the ear.24h later, all mice were euthanized by cervical dislocation and both ears were removed. Ear samples were made and swelling degree was measured. The spleen index and thymus index were also examined.
1.2.2Results
Treatment with ZL-5015(200,100and50mg/kg) suppressed the DNFB-induced ear swelling by56.45%(P＜0.001),47.58%(P＜0.001) and29.84%(P＜0.05) compared with vehicle group, respectively. There was no statistically significant effect observed on the mouse thymus index and only a decrease in spleen index was noticed at the dose of200mg/kg with statistical significance at P＜0.05.
1.3Effect of ZL-5015on phagocytosis of reticuloendothelial cells (macrophages)--Evaluated by carbon clearance.
1.3.1Materials and Methods
Fifty male and female mice were equitably and randomly assigned into5groups (n=10/group). The mice were intragastrically administered with ZL-5015for7consecutive days. After one hour of the last administration, the animals were intravenously injected (0.1ml/10g) India ink (6fold dilution with normal saline) by tail vein. The absorbance at a wavelength of450nm of the blood samples collected at the first minute and the tenth minute after the injection was read and clearance index (K) and phagocytic index(A) were calculated by correction with the weight of liver and spleen.
1.3.2Results
There was no statistically significant effect observed in mice treated with ZL-5015(200,100and50mg/kg) on phagocytic index (A)(P=1.000, P=0.999, P=1.000).
2. Evaluation of therapeutic effect on adjuvant-induced arthritis rats (AA rats)
2.1Effect of ZL-5015on arthritis index, paw edema and body mass in AA rats.
2.1.1Materials and Methods
Forty-eight female SD Rats were equitably and randomly assigned into6groups (n=8/group). The induction of adjuvant arthritis was done by intraplantar injection of100μL of Freund's complement adjuvant suspension in the rats' left hind paws. Administration of ZL-5015was started on the sixteen day when the animal exhibited the typical signs of arthritis and continued for12consecutive days. Paw volume was measured by digital plethysmography and body mass were weighed once every4days. The paw edema was expressed as the increase in paw volume after the adjuvant injection compared with the initial volume. Arthritis index was determined according to the redness and swelling of the inflamed paws after injection of Freund's complement adjuvant.
2.1.2Results
Treatment with ZL-5015(200mg/kg.b.w) decreased the arthritis index to20.0%(P=0.010) when compared with the model group on the20th day after injection, indicating that the compound started to take effect at that time. There were no obvious effect on paw edema and body mass.
On the24th day, treatment with ZL-5015(200,100and50mg/kg) decreased the arthritis index by34.5%(P=0.010),25.0%(P=0.001) and19.0%(P=0.010) compared with model groups, respectively. The high dose (200mg/kg) reduced the paw edema by49.5%(P=0.048), while middle and low doses (100and50mg/kg.b.w) slightly (no statistically) inhibited paw edema by40.4%(P=0.194) and21.5%(P=0.845), respectively. There was a slight increase in the body mass but without statistical significance (P>0.05).
On the28th day, treatment with ZL-5015(200,100and50mg/kg) decreased the arthritis index by43.0%(P=0.000),36.7%(P=0.000) and26.6%(P=0.001) compared with model groups, respectively.200and100mg/kg of ZL-5015reduced paw edema by54.0%(P=0.034) and48.7%(P=0.041), respectively, while slightly (no statistically) reducing paw edema by39.4%at doses of50mg/kg.b.w (P=0.150). There was a slight increase in the body mass but without statistical significance (P>0.05).
2.2Effect of ZL-5015on serum proinflammatory cytokines in AA rats.
2.2.1Materials and Methods
After the treatment on the last day, blood was collected from the femoral artery of the animals and centrifuged at3000rpm for10minutes to harvest the supernatant to determine the amounts of IL-1β and TNF-α by enzyme linked immunosorbent assay (ELISA).
2.2.2Results
Treatment with ZL-5015(200,100mg/kg) decreased the IL-1β production by 27.0%(P=0.006),19.1%(P=0.049), respectively, while slightly (no statistically) decreasing the IL-1β production at doses of50mg/kg.b.w (P=0.426).
Treatment with ZL-5015(200,100mg/kg) reduced the TNF-a production in a dose dependent manner with the inhibition rate for29.2%(P=0.000),21.6%(P=0.005), respectively, while slightly (no statistically) reducing the TNF-α production at doses of50mg/kg.b.w (P=0.173).
2.3Effect of ZL-5015on proinflammatory cytokines in inflamed paw of AA rats.
2.3.1Materials and Methods
After treatment on the last day, all rats were killed by decapitation. The non-injection paw was removed and cut into pieces that were then soaked in normal saline over night. The supernatants were harvested by centrifugation and the amounts of IL-1β and TNF-α in the supernatants were assessed by enzyme linked immunosorbent assay (ELISA).
2.3.2Results
Treatment with ZL-5015(200,100mg/kg) decreased the IL-1β production by30.3%(P=0.000),19.3%(P=0.019), respectively, while slightly (no statistically) decreasing the IL-1β production at doses of50mg/kg.b.w (P=0.123).
ZL-5015(200,100and50mg/kg) reduced the TNF-α production in a dose dependent manner with the inhibition rates of31.9%(P=0.000),22.0%(P=0.004) and15.2%(P=0.039), respectively.
3. Effect of ZL-5015on lymphocyte function
3.1Effect of ZL-5015on concanavalin A (Con A) and lipopolysaccharides (LPS) stimulated mouse splenocyte proliferation
3.1.1Materials and Methods
Two mice were euthanized as described before. The spleens were removed and singly splenocyte suspension was made. Cells were plated at4×105cells/well in 96-well plates and stimulated with Con A (4μg/mL) or LPS (10μg/mL) in the presence or absence of various concentrations of ZL-5015at37℃for48h. The proliferation activity of lymphocytes was measured by MTT assay.
3.1.2Results
Mouse splenocyte proliferation induced by Con A (4μg/ml) was inhibited in the presence of ZL-5015(40μM, P=0.000,20μM,P=0.000, and10μM, P=0.000) in a dose dependent manner with inhibition rates of33.1%,21.0%and13.3%, compared with Con A group, respectively.
ZL-5015(40μM, P=0.000,20μM, P=0.000and10μM, P=0.087) suppressed the splenocyte proliferation stimulated by LPS(10μg/ml) in a dose dependent manner with inhibition rates of25.90%,14.95%and4.30%, compared with LPS group, respectively
3.2Effect of ZL-5015on mixed lymphocyte culture response (MLC)
3.2.1Materials and Methods
Two mice were euthanized as described before. The spleens were removed; singly splenocyte suspension was made and mixed by1:1. Cells were plated at6×105cells/well in96-well plates in the presence or absence of various concentrations of ZL-5015at37℃for72h. The proliferation activity of lymphocytes was measured by MTT assay.
3.2.2Results
Mixed lymphocyte culture response (MLC) was inhibited in the presence of ZL-5015(40μM, P=0.000,20μM, P=0.000, and10μM, P=0.037) in a dose dependent manner with inhibition rates of37.2%,17.4%and5.0%, respectively.
3.3Effect of ZL-5015on Con A-induced production of IL-2and INF-γ by mouse splenocyte
3.3.1Materials and Methods
The singly splenocyte suspension was made as described before. Cells were plated at4×105cells/well in96-well plates and stimulated with Con A (4μg/mL) in the presence or absence of various concentrations of ZL-5015at37℃for48h. The production of IL-2and INF-y were measured by ELISA.
3.3.2Results
Con A-induced production of IL-2was inhibited in the presence of ZL-5015(40μM, P=0.000,20μM, P=0.000, and10μM, P=0.007) in a dose dependent manner with inhibition rates for29.37%,20.31%and11.35%, respectively.
Con A-induced production of INF-y was decreased in the presence of ZL-5015(40μM, P=0.000,20μM, P=0.002, and10μM, P=0.030) in a dose dependent manner with inhibition rates of31.9%,23.6%and15.9%, respectively.
3.4Effect of ZL-5015on the mRNA expression of IL-2and INF-y in mouse splenocytes induced by Con A in vitro
3.4.1Materials and Methods
The singly splenocyte suspension was made as described before. Cells were plated at6×106cells/well in6-well plates and stimulated with Con A (4μg/mL) in the presence or absence of various concentrations of ZL-5015at37℃for48h. The production of IL-2and INF-y were measured by Real time fluorescence quantitative PCR.
3.4.2Results
Con A-induced mRNA expression of IL-2was inhibited in the presence of ZL-5015(40μM, P=0.010,20μM, P=0.024, and10μM, P=0.048) in a dose dependent manner, respectively.
Con A-induced mRNA expression of INF-y was decreased in the presence of ZL-5015(40μM, P=0.005,20μM, P=0.013, and10μM, P=0.043) in a dose dependent manner, respectively.
3.5Effect of ZL-5015on the secretion of IgG1and IgG2a antibody by mouse splenocytes stimulated with LPS.
3.5.1Materials and Methods
The singly splenocyte suspension was made as described before. Cells were plated at4×105cells/well in96-well plates and stimulated with LPS (10μg/mL) in the presence or absence of various concentrations of ZL-5015at37℃for48h. The production of IgG1and IgG2a antibody were measured by ELISA.
3.5.2Results
Treatment with ZL-5015(40μM, P=0.000,20μM, P=0.001) reduced production of IgGl by27.8%and18.0%, respectively, while slightly (no statistically) reducing production of IgGl at a dose of50mg/kg.b.w (P=0.150).
LPS-induced production of IgG2a was decreased in the presence of ZL-5015(40μM, P=0.000,20μM, P=0.000, and10μM, P=0.011) in a dose dependent manner with inhibition rates of31.6%,22.7%and13.7%, respectively.
3.6Effect of ZL-5015on T lymphocyte subset
3.6.1Materials and Methods
The protocol was the same as described in section3.5.1. The cells were collected after incubation for48h and stained with fluorescein-labeled antibodies against mouse CD3, CD4and CD8antigens. After being washed with PBS, samples were immediately detected by a FACScan flow cytometer.
3.6.2Result
It was found that ZL-5015primarily reduced the proportion of CD4low and CD3low subset in mouse splenocytes activated by Con A.
Statistical Analysis
SPSS11.5statistical software was applied to statistical analysis. The proportion of T cell subsets in total cell score was expressed as percentage. The R×C crosstabs chi-square test was used for an overall test of percentage among each group, for differences among experimental groups at first, and then the four rows crosstabs chi-square test was performed for two by two comparisons. Continuous variables were described as Mean±SD and analyzed by One-way ANOVA (Analysis of Variance). Test of homogeneity of variances was carried out prior to ANOVA. If the variance was of homogeneity, LSD method was used for multiple comparison, otherwise, Dunnett's T3method was adopted for multiple comparison, using F and P values by Welch correction analysis of variance. Significance level was set at α=0.05and P＜0.05was considered to be statistical significance.
Conclusion
1. ZL-5015reduced the hemolysin level in sera induced by RRBC, suppressed the DNFB-induced ear swelling and had no statistically significant effect on carbon clearance, indicating that the compound has inhibitory effect on humoral and cellular immune function, but has no effect on the nonspecific immune function in mice.
2. ZL-5015decreased the arthritis index and paw edema in AA rats, which implies that it has the potential to be a promising drug candidate for rheumatoid arthritis (RA).
3. ZL-5015decreased the production of proinflammatory cytokines IL-1β and TNF-a both in sera and in inflamed paws of AA rats, suggesting that the therapeutic effect on adjuvant-induced arthritis rats might be related to the inhibition of proinflammatory cytokines.
4. ZL-5015suppressed the splenocyte proliferation stimulated by Con A and LPS, suggesting it can directly inhibit the proliferation of T and B lymphocytes.
5. ZL-5015significantly inhibited the cell proliferation in mixed lymphocyte culture, suggesting that ZL-5015can inhibit the lymphocyte proliferation induced by specific antigen.
6. ZL-5015significantly inhibited the production of IL-2and INF-γ induced by ConA, suggesting that the immunosuppressive action may relate to its inhibition of production and/or secretion of the cytokines.
7. ZL-5015inhibited the mRNA expression of IL-2and INF-γ induced by Con A, suggesting that the inhibition of cytokine production is resulted from the suppression of mRNA expression.
8. ZL-5015significantly inhibited the secretion of IgGl and IgG2a antibodies by lymphocytes stimulated with LPS, suggesting the compound inhibits antibody production by directly acting on B lymphocytes.
9. ZL-5015reduced the percentage of CD4low and CD3low T cells in mouse splenocytes activated by Con A, suggesting that the mechanism underlying the immunosuppressive effect might be inhibition of the activation and/or proliferation of CD4+CD3+T lymphocyte.
10. In conclusion, ZL-5015has inhibitory effect on humoral and cellular immunity, directly inhibits the proliferation and secretory function of T and B lymphocytes, and is effective to animal models of autoimmune diseases. However, the effective dose is higher (at least at100mg/kg) than that of the medicines that is being used in clinic. Thus, it needs to be further structurally modified.
新型四氢嘧啶类化合物ZL-5015(1,3-二环戊基-1,2,3,6-四氢嘧啶-4,5-、甲酸二乙酯)兼具抗炎和免疫抑制活性,本研究采用体内和体外实验模型探讨ZL-5015的免疫抑制作用特点及作用机制,为后续的结构改造及深入的作用机制研究提供参考信息。
研究内容
1.ZL-5015对正常小鼠免疫功能的影响
1.1对体液免疫功能的影响---兔红细胞免疫小鼠产生血清溶血素(IgM)模型
1.1.1材料与方法
BALB/c小鼠50只,随机分为5组,各组动物均连续7天给药,给药后第2天上午,每只鼠腹腔注射20%(V/V)的兔红细胞生理盐水0.2ml进行免疫,同日下午继续给药,免疫后第6天(免疫当天为第0天),动物摘眼球取血,分光光度法检测各组小鼠血清中的溶血素水平。
1.1.2结果
ZL-5015高、中剂量组(200.100mg/kg.b.w)能明显抑制小鼠血清溶血素水平,抑制率分别为32.8%和20.0%(P=0.000,P=0.000),ZL-5015低剂量组(50mg/kg.b.w)能轻微抑制小鼠血清溶血素水平,但与溶媒对照组比较差异无统计学意义(P=0.131)。
1.2对细胞免疫功能的影响---二硝基氟苯(DNFB)诱导小鼠迟发型超敏反应(DTH)模型
1.2.1材料与方法
BALB/c小鼠50只,随机分为5组,给药第一天用1%DNFB致敏小鼠,连续给药7天,于致敏后第7天,取1%DNFB丙酮-橄榄油(1：1)20gL均匀涂布于每只小鼠右耳的两面进行攻击。于攻击24h后,给小鼠称重,颈椎脱臼处死,剪下左右耳廓,以左右耳片质量差值作为肿胀度,并计算肿胀率。取出脾脏和胸腺称重并计算相应的器官指数。
1.2.2结果
ZL-5015高、中、低剂量组(200、100、50mg/kg.b.w)与模型组比较,均能显著抑制二硝基氟苯诱导的小鼠耳廓肿胀(P=0.000,P=0.000,P=0.015),抑制率分别为：56.45%、47.58%、29.84%。ZL-5015高剂量(200mg/kg.b.w)能抑制小鼠的脾脏指数(P=0.013),中、低剂量组(100、50mg/kg.b.w)无抑制作用(P=0.482,P=0.418)。ZL-5015高、中、低剂量组(200、100.50mg/kg.b.w)对小鼠的胸腺指数无显著影响(P=0.384,P=0.527,P=0.570)。
1.3ZL-5015对网状内皮细胞(巨噬细胞)吞噬功能的影响(碳粒廓清实验)
1.3.1材料与方法
BALB/c小鼠50只,随机分为5组,连续给药7天。末次给药1h后,先称小鼠体重,每只小鼠尾静脉均注射印度墨汁(用生理盐水稀释6倍),按0.1ml/10g注射。分别于注射后于1min(t1)和10min(t10)小鼠眼球取血,检测OD450值,最后将小鼠颈椎脱臼处死,同时摘取小鼠肝脏、脾脏,并称湿重,计算廓清指数(K)及吞噬指数(A)。
1.3.2结果
化合物ZL-5015高、中、低剂量组(200、100、50mg/kg.b.w)对小鼠吞噬指数A无明显影响(P=1.000,P=0.999,P=1.000)。
2.ZL-5015对大鼠佐剂性关节炎模型的影响
2.1ZL-5015对大鼠佐剂性关节炎模型关节炎指数、足趾肿胀度、体重的影响
2.1.1材料与方法
雌性SD大鼠48只,随机分为6组,每只鼠左后足趾皮内注射100μL弗氏佐剂,致炎后第16天开始给药和测量,共给药12天。采用排水法测足部体积：在大鼠足部踝骨关节突出部位划一条线作为标记,用足趾肿胀仪测量,间隔4天测一次足趾肿胀情况及体重。SD大鼠免疫后,根据大鼠四肢关节炎症的红、肿及硬化程度,计算多发性关节炎指数。
2.1.2结果
致炎后第20天,ZL-5015高剂量(200mg/kg.b.w)处理组关节炎指数与模型组比较明显下降,下降率为20.0%(P=0.010),说明此时高剂量药物开始起效。对足趾肿胀及体重无显著影响。
致炎后第24天,ZL-5015高、中、低剂量(200、100、50mg/kg.b.w)使关节炎指数分别下降34.5%、25.0%、19.0%(P=0.000,P=0.001,P=0.010)。高剂量能使足趾肿胀度下降49.5%(P=0.048),中、低剂量使足趾肿胀度分别下降40.4%及21.5%,但无统计学意义(P=0.194,P=0.845)。对体重有增重趋势,但差异无统计学意义。
致炎后第28天,ZL-5015高、中、低剂量(200、100、50mg/kg.b.w)使关节炎指数分别下降43.0%、36.7%、26.6(P=0.000,P=0.000,P=0.001)。高、中剂量使足趾肿胀分别下降54.0%及48.7%(P=0.034,P=0.041),低剂量下降39.4%,但差异无统计学意义(P=0.150)。对体重有增重趋势,但差异无统计学意义。
2.2ZL-5015对弗氏佐剂关节炎大鼠血清促炎细胞因子的影响
2.2.1材料与方法
于佐剂关节炎造模第28天,股动脉取血,3000rpm离心10min,取上清,ELISA方法检测IL-1β及TNF-α活性
2.2.2结果
ZL-5015高、中剂量(200、100mg/kg)与模型组比较,均可显著降低关节炎大鼠血清IL-1β的水平(P=0.006,P=0.049),且有浓度依赖趋势,抑制率分别为：27.0%,19.1%；ZL-5015低剂量组(50mg/kg)可以轻微降低大鼠血清IL-1β的水平(P=0.426)。ZL-5015高、中剂量组(200、100mg/kg)均可以显著降低大鼠血清TNF-a的水平(P=0.000,P=0.005),且有浓度依赖趋势,抑制率分别为：29.2%,21.6%；ZL-5015低剂量组(50mg/kg可以轻微降低大鼠血清TNF-α的水平,但差异无统计学意义(P=0.173)。
2.3ZL-5015对关节炎部位促炎细胞因子的影响
2.3.1材料与方法
于造模第28天,大鼠断头处死,在非致炎侧踝关节上方0.5cm处摘取肿胀的足爪,剪碎,生理盐水浸泡过夜,离心取上清,ELISA方法检测IL-1β及TNF-a活性。
2.3.2结果
ZL-5015高、中剂量(200、100mg/kg)与模型组比较,均可显著减少关节炎部位IL-1β的含量(P=0.000,P=0.019),且有浓度依赖趋势,抑制率分别为：30.3%,19.3%；ZL-5015低剂量(50mg/kg)可以轻微减少炎症部位IL-1β的含量,但差异无统计学意义(P=0.123)。ZL-5015高、中、低剂量组(200、100、50mg/kg)均可显著减少炎症部位TNF-α的含量(P=0.000,P=0.004,P=0.039),且有浓度依赖趋势,抑制率分别为：31.9%,22.0%和15.2%。
3.ZL-5015对淋巴细胞功能的影响
3.1ZL-5015对Con A和LPS诱导小鼠脾细胞增殖反应的影响
3.1.1材料与方法
Balb/c小鼠两只,常规制备脾细胞悬液,Con A(4μg/ml)和LPS(10μg/ml)刺激小鼠脾细胞增殖作为体外免疫模型,检测给药48小时后各组细胞的代谢活力。
3.1.2结果
ZL-5015高、中、低剂量均可以显著抑制Con A诱导的小鼠脾细胞增殖反应,(P=0.000,P=0.000,P=0.000),且有浓度依赖趋势,抑制率分别是：33.10%、21.0%和13.28%。ZL-5015高、中剂量组均可以显著抑制LPS诱导的小鼠脾细胞增殖反应(P=0.000,P=0.000),且有浓度依赖趋势,抑制率分别是：25.90%、14.95%,ZL-5015低剂量组有轻微抑制LPS诱导的小鼠脾细胞增殖反应的趋势,但差异无统计学意义(P=0.087)。
3.2ZL-5015对混合淋巴细胞培养反应的影响
3.2.1材料与方法
将BALB/c小鼠和C57BL/6J小鼠的脾细胞分别制成悬液,按照1：1混合制成细胞浓度为6x106个/mL的细胞悬液,按分组加入药物,用96孔培养板培养72h后各组细胞的代谢活力。
3.2.2结果
ZL-5015高、中、低剂量组均可以显著抑制混合淋巴细胞的增殖反应(P=0.000,P=0.000,P=0.037),且有浓度依赖趋势,抑制率分别是：37.2%、17.4%和5.0%。
3.3ZL-5015对Con A刺激小鼠脾细胞分泌白介素2和干扰素γ的影响
3.3.1材料与方法
Balb/c小鼠两只,常规制备脾细胞悬液,Con A(4μg/ml)刺激小鼠脾细胞活化,检测给药48小时后各组细胞中上清液白细胞介素2和干扰素γ的含量。
3.3.2结果
ZL-5015高、中、低剂量组(40、20、10μM)均可显著抑制Con A诱导的小鼠脾细胞分泌白介素2(P=0.000,P=0.000,P=0.007),且有浓度依赖趋势,抑制率分别是：29.37%、20.31%和11.35%。ZL-5015高、中、低剂量组(40、20、10μM)均可以显著抑制Con A诱导的小鼠脾细胞分泌干扰素γ(P=0.000,P=0.002,P=0.030),且有浓度依赖趋势,抑制率分别是：31.9%、23.6%和15.9%。
3.4ZL-5015对小鼠脾细胞细胞因子mRNA表达的抑制作用
3.4.1材料与方法
Balb/c小鼠两只,常规制备脾细胞悬液,Con A(4μg/ml)刺激小鼠脾细胞活化,使用荧光定量PCR检测给药48小时后各组细胞中白细胞介素2和干扰素γmRNA表达的变化。
3.4.2结果
ZL-5015(40.20.10μM)可抑制由Con A诱导的IL-2mRNA的表达(P=0.010,P=0.024,P=0.048)。ZL-5015(40、20、10μM)可抑制由Con A诱导的INF-γmRNA的表达(P=0.005,P=0.013,P=0.043)。
3.5ZL-5015对LPS刺激小鼠脾细胞分泌IgG1和IgG2a的影响
3.5.1材料与方法
Balb/c小鼠两只,常规制备脾细胞悬液,LPS(10μg/ml)激小鼠脾细胞活化,各组给药作用48小时后,收集培养上清液检测IgGl和IgG2a的含量。
3.5.2结果
ZL-5015高、中剂量组(40、20μM)均可以显著抑制LPS诱导的小鼠脾细胞分泌IgGl,(P=0.000,P=0.001),且有浓度依赖趋势,抑制率分别是：27.8%、18.0%；ZL-5015低剂量组(10μM)可以轻微抑制LPS诱导的小鼠脾细胞分泌IgGl,但差异无统计学意义(P=0.105)。ZL-5015高、中、低剂量组(40、20、10μM)均可以显著抑制LPS诱导的小鼠脾细胞分泌IgG2a(P=0.000,P=0.000,P=0.011),且有浓度依赖趋势,抑制率分别是：31.6%、22.7%和13.7%。
3.6ZL-5015对T细胞亚类的影响
3.6.1材料与方法
脾细胞制备、实验分组及给药均同3.5.1所述,48小时后收集细胞,用荧光标记的抗小鼠CD3、CD4、 CD8抗体染色,流式细胞仪检测。统计方法
3.6.2结果
ZL-5015主要降低Con A诱导的CD4弱表达和CD3弱表达T细胞亚群(CD4LowCD3Low)的比例。
统计学处理
应用SPSS11.5统计软件进行统计分析。T细胞亚类占细胞总数的比例以百分数表示,先采用RxC表资料的卡方检验对各组细胞的百分率差异进行多组间的总体检验,然后采用四格表资料的卡方检验进行组间细胞百分率的两两比较。定量数据结果以均数±标准差表示,采用单向方差分析法分析,先进行方差齐性检验,若方差齐用LSD方法多重比较；若方差不齐,采用Welch F检验和Dunnett's T3法多重比较。显著性水准取α=0.05,以P<0.05时,组间差异具有统计学意义。
结论
1、ZL-5015可降低兔红细胞诱导小鼠产生血清溶血素的活性,降低二硝基氟苯诱导的小鼠耳廓的肿胀度,对小鼠碳粒廓清的速率无明显影响,提示对小鼠的体液免疫及细胞免疫功能有抑制作用,对非特异性免疫功能无影响。
2、ZL-5015可以减轻弗氏佐剂关节炎模型的关节炎指数,足趾肿胀,提示对类风湿关节炎具有一定的临床应用前景。
3、ZL-5015可以减少弗氏佐剂关节炎模型血清及炎症部位中的促炎细胞因子IL-1β和TNF-α的含量,提示对佐剂性关节炎的治疗作用与抑制促炎细胞因子的产生与关。
4、ZL-5015可抑制Con A和LPS诱导的小鼠脾细胞的增殖反应,提示对T淋巴细胞和B淋巴细胞的增殖反应有直接的抑制作用。
5、ZL-5015可以显著抑制混合淋巴细胞的增殖反应(代谢活力),提示对特异抗原诱导的淋巴细胞增殖反应有直接的抑制作用。
6、ZL-5015可以显著抑制Con A诱导的小鼠脾细胞分泌白介素2和干扰素γ,提示其免疫抑制作用与抑制细胞因子的产生与分泌有关。
7、ZL-5015可抑制由Con A诱导的IL-2及INF-y mRNA的表达,提示其抑制细胞因子产生的作用环节在mRNA表达水平。
8、ZL-5015可以显著抑制LPS诱导的小鼠脾细胞分泌IgGl和IgG2a,提示其可直接作用于B淋巴细胞而抑制抗体的产生。
9、ZL-5015可降低Con A诱导的CD4弱表达和CD3弱表达T细胞亚群(CD4LowCD3Low)的比例,提示其作用机制可能是抑制CD4+CD3+细胞的活化与增殖,从而抑制免疫反应。
10、总之,ZL-5015对体液和细胞免疫反应均有抑制作用,对T和B淋巴细胞的增殖与分泌功能具有直接抑制作用,对自身免疫性疾病动物模型有效,但需要高剂量(100mg/kg以上),与现有临床药物相比其作用强度还不够理想,需要进一步结构改造。
Objective
The novel tetrahydropyrimidine compound ZL-5015(1,3-dicyclopentyl-1,2,3,6-tetrahydropyrimidine-4,5-dicarboxylic acid diethyl ester) possesses both anti-inflammatory and immune suppressive activities. This study was undertaken to investigate its immunosuppressive characteristics and the underlying mechanisms in vivo and in vitro in an attempt to provide relevant information for further structure modification and research.
Research content
1. Effect of ZL-5015on mouse immune function
1.1Effect of ZL-5015on humoral immune function---Evaluated by the level of serum hemolysin (IgM) in mice immunized with rabbit red blood cells.
1.1.1Materials and Methods
Fifty male and female mice were equitably and randomly assigned into5groups (n=10/group). The mice were intragastrically administered with ZL-5015for7consecutive days. On the second day, the animals were primed with0.2mL rabbit red blood cells (RRBC,20%v/v, per mouse, i.p.). After the treatment on the last day, blood was collected from the fossa orbitalis plexus venosus of the animals and the level of hemolysin was determined by spectrophotometry.
1.1.2Results
ZL-5015(200,100and50mg/kg) reduced the hemolysin level in sera in mice induced by RRBC in a dose dependent manner with the inhibition rates of32.8%(P＜0.001),20.0%(P＜0.001), and8.1%(P＞0.05), respectively.
1.2Effect of ZL-5015on cellular immune function---Evaluated by delayed type hypersensitivity (DTH) induced by DNFB
1.2.1Materials and Methods
Fifty male and female mice were equitably and randomly assigned into5groups (n=10/group).On the first day after intragastric administration of ZL-5015, the mice were primed with DNFB by applying20μL1%DNFB in1:1acetone-olive oil to the hair removed area of the animal abdomen (2cm×2cm). ZL-5015was administered once a day for7consecutive days. On the sixth day,20μL1%DNFB solution was used to challenge the left ear of the mice by applying evenly to the dorsal and ventral surfaces of the ear.24h later, all mice were euthanized by cervical dislocation and both ears were removed. Ear samples were made and swelling degree was measured. The spleen index and thymus index were also examined.
1.2.2Results
Treatment with ZL-5015(200,100and50mg/kg) suppressed the DNFB-induced ear swelling by56.45%(P＜0.001),47.58%(P＜0.001) and29.84%(P＜0.05) compared with vehicle group, respectively. There was no statistically significant effect observed on the mouse thymus index and only a decrease in spleen index was noticed at the dose of200mg/kg with statistical significance at P＜0.05.
1.3Effect of ZL-5015on phagocytosis of reticuloendothelial cells (macrophages)--Evaluated by carbon clearance.
1.3.1Materials and Methods
Fifty male and female mice were equitably and randomly assigned into5groups (n=10/group). The mice were intragastrically administered with ZL-5015for7consecutive days. After one hour of the last administration, the animals were intravenously injected (0.1ml/10g) India ink (6fold dilution with normal saline) by tail vein. The absorbance at a wavelength of450nm of the blood samples collected at the first minute and the tenth minute after the injection was read and clearance index (K) and phagocytic index(A) were calculated by correction with the weight of liver and spleen.
1.3.2Results
There was no statistically significant effect observed in mice treated with ZL-5015(200,100and50mg/kg) on phagocytic index (A)(P=1.000, P=0.999, P=1.000).
2. Evaluation of therapeutic effect on adjuvant-induced arthritis rats (AA rats)
2.1Effect of ZL-5015on arthritis index, paw edema and body mass in AA rats.
2.1.1Materials and Methods
Forty-eight female SD Rats were equitably and randomly assigned into6groups (n=8/group). The induction of adjuvant arthritis was done by intraplantar injection of100μL of Freund's complement adjuvant suspension in the rats' left hind paws. Administration of ZL-5015was started on the sixteen day when the animal exhibited the typical signs of arthritis and continued for12consecutive days. Paw volume was measured by digital plethysmography and body mass were weighed once every4days. The paw edema was expressed as the increase in paw volume after the adjuvant injection compared with the initial volume. Arthritis index was determined according to the redness and swelling of the inflamed paws after injection of Freund's complement adjuvant.
2.1.2Results
Treatment with ZL-5015(200mg/kg.b.w) decreased the arthritis index to20.0%(P=0.010) when compared with the model group on the20th day after injection, indicating that the compound started to take effect at that time. There were no obvious effect on paw edema and body mass.
On the24th day, treatment with ZL-5015(200,100and50mg/kg) decreased the arthritis index by34.5%(P=0.010),25.0%(P=0.001) and19.0%(P=0.010) compared with model groups, respectively. The high dose (200mg/kg) reduced the paw edema by49.5%(P=0.048), while middle and low doses (100and50mg/kg.b.w) slightly (no statistically) inhibited paw edema by40.4%(P=0.194) and21.5%(P=0.845), respectively. There was a slight increase in the body mass but without statistical significance (P>0.05).
On the28th day, treatment with ZL-5015(200,100and50mg/kg) decreased the arthritis index by43.0%(P=0.000),36.7%(P=0.000) and26.6%(P=0.001) compared with model groups, respectively.200and100mg/kg of ZL-5015reduced paw edema by54.0%(P=0.034) and48.7%(P=0.041), respectively, while slightly (no statistically) reducing paw edema by39.4%at doses of50mg/kg.b.w (P=0.150). There was a slight increase in the body mass but without statistical significance (P>0.05).
2.2Effect of ZL-5015on serum proinflammatory cytokines in AA rats.
2.2.1Materials and Methods
After the treatment on the last day, blood was collected from the femoral artery of the animals and centrifuged at3000rpm for10minutes to harvest the supernatant to determine the amounts of IL-1β and TNF-α by enzyme linked immunosorbent assay (ELISA).
2.2.2Results
Treatment with ZL-5015(200,100mg/kg) decreased the IL-1β production by 27.0%(P=0.006),19.1%(P=0.049), respectively, while slightly (no statistically) decreasing the IL-1β production at doses of50mg/kg.b.w (P=0.426).
Treatment with ZL-5015(200,100mg/kg) reduced the TNF-a production in a dose dependent manner with the inhibition rate for29.2%(P=0.000),21.6%(P=0.005), respectively, while slightly (no statistically) reducing the TNF-α production at doses of50mg/kg.b.w (P=0.173).
2.3Effect of ZL-5015on proinflammatory cytokines in inflamed paw of AA rats.
2.3.1Materials and Methods
After treatment on the last day, all rats were killed by decapitation. The non-injection paw was removed and cut into pieces that were then soaked in normal saline over night. The supernatants were harvested by centrifugation and the amounts of IL-1β and TNF-α in the supernatants were assessed by enzyme linked immunosorbent assay (ELISA).
2.3.2Results
Treatment with ZL-5015(200,100mg/kg) decreased the IL-1β production by30.3%(P=0.000),19.3%(P=0.019), respectively, while slightly (no statistically) decreasing the IL-1β production at doses of50mg/kg.b.w (P=0.123).
ZL-5015(200,100and50mg/kg) reduced the TNF-α production in a dose dependent manner with the inhibition rates of31.9%(P=0.000),22.0%(P=0.004) and15.2%(P=0.039), respectively.
3. Effect of ZL-5015on lymphocyte function
3.1Effect of ZL-5015on concanavalin A (Con A) and lipopolysaccharides (LPS) stimulated mouse splenocyte proliferation
3.1.1Materials and Methods
Two mice were euthanized as described before. The spleens were removed and singly splenocyte suspension was made. Cells were plated at4×105cells/well in 96-well plates and stimulated with Con A (4μg/mL) or LPS (10μg/mL) in the presence or absence of various concentrations of ZL-5015at37℃for48h. The proliferation activity of lymphocytes was measured by MTT assay.
3.1.2Results
Mouse splenocyte proliferation induced by Con A (4μg/ml) was inhibited in the presence of ZL-5015(40μM, P=0.000,20μM,P=0.000, and10μM, P=0.000) in a dose dependent manner with inhibition rates of33.1%,21.0%and13.3%, compared with Con A group, respectively.
ZL-5015(40μM, P=0.000,20μM, P=0.000and10μM, P=0.087) suppressed the splenocyte proliferation stimulated by LPS(10μg/ml) in a dose dependent manner with inhibition rates of25.90%,14.95%and4.30%, compared with LPS group, respectively
3.2Effect of ZL-5015on mixed lymphocyte culture response (MLC)
3.2.1Materials and Methods
Two mice were euthanized as described before. The spleens were removed; singly splenocyte suspension was made and mixed by1:1. Cells were plated at6×105cells/well in96-well plates in the presence or absence of various concentrations of ZL-5015at37℃for72h. The proliferation activity of lymphocytes was measured by MTT assay.
3.2.2Results
Mixed lymphocyte culture response (MLC) was inhibited in the presence of ZL-5015(40μM, P=0.000,20μM, P=0.000, and10μM, P=0.037) in a dose dependent manner with inhibition rates of37.2%,17.4%and5.0%, respectively.
3.3Effect of ZL-5015on Con A-induced production of IL-2and INF-γ by mouse splenocyte
3.3.1Materials and Methods
The singly splenocyte suspension was made as described before. Cells were plated at4×105cells/well in96-well plates and stimulated with Con A (4μg/mL) in the presence or absence of various concentrations of ZL-5015at37℃for48h. The production of IL-2and INF-y were measured by ELISA.
3.3.2Results
Con A-induced production of IL-2was inhibited in the presence of ZL-5015(40μM, P=0.000,20μM, P=0.000, and10μM, P=0.007) in a dose dependent manner with inhibition rates for29.37%,20.31%and11.35%, respectively.
Con A-induced production of INF-y was decreased in the presence of ZL-5015(40μM, P=0.000,20μM, P=0.002, and10μM, P=0.030) in a dose dependent manner with inhibition rates of31.9%,23.6%and15.9%, respectively.
3.4Effect of ZL-5015on the mRNA expression of IL-2and INF-y in mouse splenocytes induced by Con A in vitro
3.4.1Materials and Methods
The singly splenocyte suspension was made as described before. Cells were plated at6×106cells/well in6-well plates and stimulated with Con A (4μg/mL) in the presence or absence of various concentrations of ZL-5015at37℃for48h. The production of IL-2and INF-y were measured by Real time fluorescence quantitative PCR.
3.4.2Results
Con A-induced mRNA expression of IL-2was inhibited in the presence of ZL-5015(40μM, P=0.010,20μM, P=0.024, and10μM, P=0.048) in a dose dependent manner, respectively.
Con A-induced mRNA expression of INF-y was decreased in the presence of ZL-5015(40μM, P=0.005,20μM, P=0.013, and10μM, P=0.043) in a dose dependent manner, respectively.
3.5Effect of ZL-5015on the secretion of IgG1and IgG2a antibody by mouse splenocytes stimulated with LPS.
3.5.1Materials and Methods
The singly splenocyte suspension was made as described before. Cells were plated at4×105cells/well in96-well plates and stimulated with LPS (10μg/mL) in the presence or absence of various concentrations of ZL-5015at37℃for48h. The production of IgG1and IgG2a antibody were measured by ELISA.
3.5.2Results
Treatment with ZL-5015(40μM, P=0.000,20μM, P=0.001) reduced production of IgGl by27.8%and18.0%, respectively, while slightly (no statistically) reducing production of IgGl at a dose of50mg/kg.b.w (P=0.150).
LPS-induced production of IgG2a was decreased in the presence of ZL-5015(40μM, P=0.000,20μM, P=0.000, and10μM, P=0.011) in a dose dependent manner with inhibition rates of31.6%,22.7%and13.7%, respectively.
3.6Effect of ZL-5015on T lymphocyte subset
3.6.1Materials and Methods
The protocol was the same as described in section3.5.1. The cells were collected after incubation for48h and stained with fluorescein-labeled antibodies against mouse CD3, CD4and CD8antigens. After being washed with PBS, samples were immediately detected by a FACScan flow cytometer.
3.6.2Result
It was found that ZL-5015primarily reduced the proportion of CD4low and CD3low subset in mouse splenocytes activated by Con A.
Statistical Analysis
SPSS11.5statistical software was applied to statistical analysis. The proportion of T cell subsets in total cell score was expressed as percentage. The R×C crosstabs chi-square test was used for an overall test of percentage among each group, for differences among experimental groups at first, and then the four rows crosstabs chi-square test was performed for two by two comparisons. Continuous variables were described as Mean±SD and analyzed by One-way ANOVA (Analysis of Variance). Test of homogeneity of variances was carried out prior to ANOVA. If the variance was of homogeneity, LSD method was used for multiple comparison, otherwise, Dunnett's T3method was adopted for multiple comparison, using F and P values by Welch correction analysis of variance. Significance level was set at α=0.05and P＜0.05was considered to be statistical significance.
Conclusion
1. ZL-5015reduced the hemolysin level in sera induced by RRBC, suppressed the DNFB-induced ear swelling and had no statistically significant effect on carbon clearance, indicating that the compound has inhibitory effect on humoral and cellular immune function, but has no effect on the nonspecific immune function in mice.
2. ZL-5015decreased the arthritis index and paw edema in AA rats, which implies that it has the potential to be a promising drug candidate for rheumatoid arthritis (RA).
3. ZL-5015decreased the production of proinflammatory cytokines IL-1β and TNF-a both in sera and in inflamed paws of AA rats, suggesting that the therapeutic effect on adjuvant-induced arthritis rats might be related to the inhibition of proinflammatory cytokines.
4. ZL-5015suppressed the splenocyte proliferation stimulated by Con A and LPS, suggesting it can directly inhibit the proliferation of T and B lymphocytes.
5. ZL-5015significantly inhibited the cell proliferation in mixed lymphocyte culture, suggesting that ZL-5015can inhibit the lymphocyte proliferation induced by specific antigen.
6. ZL-5015significantly inhibited the production of IL-2and INF-γ induced by ConA, suggesting that the immunosuppressive action may relate to its inhibition of production and/or secretion of the cytokines.
7. ZL-5015inhibited the mRNA expression of IL-2and INF-γ induced by Con A, suggesting that the inhibition of cytokine production is resulted from the suppression of mRNA expression.
8. ZL-5015significantly inhibited the secretion of IgGl and IgG2a antibodies by lymphocytes stimulated with LPS, suggesting the compound inhibits antibody production by directly acting on B lymphocytes.
9. ZL-5015reduced the percentage of CD4low and CD3low T cells in mouse splenocytes activated by Con A, suggesting that the mechanism underlying the immunosuppressive effect might be inhibition of the activation and/or proliferation of CD4+CD3+T lymphocyte.
10. In conclusion, ZL-5015has inhibitory effect on humoral and cellular immunity, directly inhibits the proliferation and secretory function of T and B lymphocytes, and is effective to animal models of autoimmune diseases. However, the effective dose is higher (at least at100mg/kg) than that of the medicines that is being used in clinic. Thus, it needs to be further structurally modified.
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