罗格列酮改善脂多糖诱导慢性低度炎症大鼠的卵巢功能异常

引用本文

俞晓敏, 闻笔伟, 张俊洁. 罗格列酮改善脂多糖诱导慢性低度炎症大鼠的卵巢功能异常[J]. 第二军医大学学报, 2019, 40(12): 1310-1316

YU Xiao-min, WEN Bi-wei, ZHANG Jun-jie. Rosiglitazone improves ovarian dysfunction of rats with chronic low-grade inflammation induced by lipopolysaccharide[J]. Academic Journal of Second Military Medical University, 2019, 40(12): 1310-1316 (in Chinese with English abstract)

罗格列酮改善脂多糖诱导慢性低度炎症大鼠的卵巢功能异常

俞晓敏^Δ, 闻笔伟^Δ, 张俊洁

海军军医大学(第二军医大学)长海医院妇产科, 上海 200433

收稿日期: 2019-08-03 接受日期: 2019-10-28

基金项目: 国家自然科学基金（81571387）.

作者简介: 俞晓敏, 硕士, 住院医师.E-mail:yuxiaomin0211@163.com;
闻笔伟, 硕士, 住院医师.E-mail:wenbiwei1990@163.com.

通信作者(Corresponding author): 张俊洁.Tel:021-31162044, E-mail:zhangjj910@163.com.

^△共同第一作者(Co-first authors)

摘要: 目的观察慢性低度炎症大鼠卵巢组织中过氧化物酶体增殖物激活受体（PPAR）的表达，并初步探究PPAR-γ激动剂罗格列酮对慢性低度炎症诱导的卵巢功能异常的影响。方法以腹腔注射脂多糖（LPS）诱导的慢性低度炎症大鼠为动物模型。将200只大鼠随机分为对照组（NS组）和慢性低度炎症组（LPS组），每组100只，分别予腹腔注射生理盐水和LPS干预。在发情周期不同阶段（发情前期、发情期、发情后期和发情间期），通过检测2组大鼠血清中雌二醇（E2）、卵泡刺激素（FSH）、黄体生成素（LH）和抗苗勒管激素（AMH）评估卵巢功能，利用蛋白质印迹法检测卵巢组织中PPAR-α、PPAR-δ和PPAR-γ的表达情况。将2组各80只大鼠分别随机分为2个亚组，每个亚组40只大鼠，分别予生理盐水和罗格列酮灌胃干预。灌胃14 d后，观察各亚组大鼠在发情周期不同阶段卵巢组织中促炎细胞因子[白细胞介素（IL）-1β、IL-6和肿瘤坏死因子α（TNF-α）]水平和卵巢功能。结果与NS组相比，在发情周期不同阶段，LPS组大鼠血清中E2和AMH水平均下降（P均 < 0.05），FSH和LH水平均升高（P均 < 0.05）；卵巢组织中PPAR-γ表达均降低（P均 < 0.05），而PPAR-α和PPAR-δ表达无明显变化（P均>0.05）。在发情周期不同阶段，LPS诱导的慢性低度炎症大鼠在口服罗格列酮后卵巢组织中促炎细胞因子IL-1β、IL-6和TNF-α水平均降低（P均 < 0.05），并且LPS诱导血清中降低的E2、AMH和升高的FSH、LH均得以改善（P均 < 0.05）。结论 PPAR-γ激动剂罗格列酮可减轻LPS诱导的大鼠慢性低度炎症反应，并改善卵巢功能。

关键词: 过氧化物酶体增殖物激活受体罗格列酮慢性低度炎症脂多糖卵巢功能异常

Rosiglitazone improves ovarian dysfunction of rats with chronic low-grade inflammation induced by lipopolysaccharide

YU Xiao-min^Δ, WEN Bi-wei^Δ, ZHANG Jun-jie

Department of Obstetrics and Gynecology, Changhai Hospital, Naval Medical University (Second Military Medical University), Shanghai 200433, China

Supported by National Natural Science Foundation of China (81571387).

Abstract: Objective To detect the expression of peroxisome proliferator-activated receptor (PPAR) in ovarian tissues of rats with chronic low-grade inflammation, and to explore the effect of PPAR-γ agonist rosiglitazone on ovarian dysfunction induced by chronic low-grade inflammation. Methods A chronic low-grade inflammation model of rats was established by intraperitoneal injection of lipopolysaccharide (LPS). Two hundred rats were randomly assigned to control group (NS group) and chronic low-grade inflammation group (LPS group), and the rats were intraperitoneally injected with normal saline and LPS, respectively. The ovarian function of rats was assessed by detecting the serum levels of estradiol (E2), follicle stimulating hormone (FSH), luteinizing hormone (LH), and anti-Müllerian hormone (AMH) during different stages of the estrus cycle (proestrus, estrus, metestrus and diestrus). The protein expression levels of PPAR-α, PPAR-δ and PPAR-γ in ovarian tissues were detected using Western blotting. Eighty rats of each group were randomly divided into two subgroups, which were administered intragastrically by normal saline and rosiglitazone, respectively. Fourteen days after intragastric administration, the levels of pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6 and tumor necrosis factor α (TNF-α), in ovarian tissues and ovarian function of rats in each subgroup were observed during different stages of the estrus cycle. Results Compared with the NS group, during different stages of the estrus cycle, the serum levels of E2 and AMH of rats in the LPS group were significantly decreased (all P < 0.05), while the serum levels of FSH and LH were significantly increased (all P < 0.05). During different stages of the estrus cycle, the expression levels of PPAR-γ in ovarian tissues were significantly decreased in the LPS group compared with the NS group (all P < 0.05), while the expression levels of PPAR-α and PPAR-δ were not significantly different between the two groups (all P>0.05). Compared with the intraperitoneal injection of LPS+intragastric administration of normal saline subgroup, during different stages of the estrus cycle, the expression levels IL-1β, IL-6 and TNF-α in ovarian tissues of rats were significantly decreased in the intraperitoneal injection of LPS+intragastric administration of rosiglitazone subgroup (all P < 0.05), the serum levels of E2 and AMH were significantly increased (all P < 0.05), and the serum levels of FSH and LH were significantly decreased (all P < 0.05). Conclusion PPAR-γ agonist rosiglitazone can attenuate LPS-induced chronic low-grade inflammatory and improve ovarian function in rats.

Key words: peroxisome proliferator-activated receptor rosiglitazone chronic low-grade inflammation lipopolysaccharide ovarian dysfunction

卵巢作为女性的主性器官，具有产生卵细胞和分泌性激素的生理功能，其功能异常严重威胁女性生殖健康^[1]。越来越多的证据表明慢性低度炎症在卵巢癌、多囊卵巢综合征等卵巢功能障碍疾病的发生、发展中起关键作用^[2-4]，但其发病机制错综复杂，慢性低度炎症与卵巢功能异常之间的关系并未完全阐明。研究表明，脂多糖(lipopolysaccharide，LPS)可抑制卵泡膜细胞类固醇激素的产生^[5]，全身低度炎症可导致卵泡生长过早停止和女性不孕^[6-7]，提示抑制慢性低度炎症可改善卵巢功能。过氧化物酶体增殖物激活受体(peroxisome proliferator-activated receptor，PPAR)是一组核受体蛋白，主要包括α、δ和γ 3种亚型，作为转录因子通过调控基因表达^[8]在细胞分化、发育和能量代谢中起重要作用^[9-10]。研究证实PPAR-α和PPAR-γ被激活后均可降低肺泡上皮细胞中LPS诱导的炎症反应^[11-12]，激活PPAR-γ可通过抑制核因子κB通路减轻LPS诱导的腹膜炎^[13]，表明激活PPAR可减轻炎症反应。本研究利用LPS诱导慢性低度炎症大鼠模型，观察慢性低度炎症对卵巢组织中PPAR表达的影响，并初步探究PPAR-γ激动剂罗格列酮对慢性低度炎症诱导的卵巢功能异常的效应，以期寻找一种保护卵巢功能的新途径。

1 材料和方法 1.1 慢性低度炎症大鼠模型的建立与分组设计

180~250 g健康雌性SD大鼠购自上海西普尔-必凯实验动物有限公司[实验动物生产许可证号SCXK(沪)-2017-0002]。适应性饲养1周后，将大鼠随机分为对照组(NS组)和慢性低度炎症组(LPS组)。NS组(n = 100)大鼠连续14 d腹腔注射生理盐水(2 mL·kg^-1·d^-1)。LPS组大鼠(n = 100)连续14 d腹腔注射溶于生理盐水的LPS(1.2 mg·kg^-1·d^-1)诱导慢性低度全身性炎症，LPS注射剂量参考Wu等^[14]的研究。每组取20只大鼠用于检测处于发情周期不同阶段大鼠血清中雌二醇(estradiol，E2)、卵泡刺激素(follicle stimulating hormone，FSH)、黄体生成素(luteinizing hormone，LH)的浓度，并随机检测其中各组10只大鼠的血清抗苗勒管激素(anti-Müllerian hormone，AMH)浓度及各组5只大鼠卵巢组织中PPAR的表达情况。其余大鼠用于罗格列酮试验。将2组其余80只大鼠按照体质量依次编号后，采用随机数表法随机分为2个亚组，分别为溶剂对照亚组(NS + Veh、LPS + Veh)和罗格列酮亚组(NS + RSG、LPS + RSG)，每个亚组40只大鼠。各亚组随机取20只大鼠，检测处于发情周期不同阶段大鼠卵巢组织中促炎细胞因子水平及血清中E2、FSH和LH的浓度。同时检测各亚组所有大鼠血清中AMH的浓度。

1.2 口服罗格列酮治疗

NS + Veh、NS + RSG亚组的大鼠在腹腔注射生理盐水后，分别接受生理盐水(4 mL·kg^-1·d^-1)灌胃和罗格列酮(20 mg·kg^-1·d^-1)灌胃^[15]。LPS + Veh、LPS + RSG亚组的大鼠在腹腔注射LPS后，也分别接受生理盐水(4 mL·kg^-1·d^-1)灌胃和罗格列酮(20 mg·kg^-1·d^-1)灌胃。腹腔注射和灌胃均为每天1次，连续干预14 d。

1.3 样本收集

雌性大鼠发情周期为4~5 d，包括4个阶段：发情前期、发情期、发情后期和发情间期，发情周期大鼠血中激素水平会有波动^[16]，因此，取血前对大鼠进行阴道细胞学检查，以确定每只大鼠所处的发情周期阶段，最终使处于发情周期4个阶段的大鼠数量相等。大鼠腹腔注射2%戊巴比妥钠3 mL/kg麻醉后，股静脉插管，取2 mL血液至4 mL离心管中，室温静置10 min，2 000 r/min(离心半径为7 cm)离心20 min，收集上清，置于- 80 ℃保存。取血后大鼠经过量麻醉处死，立即取出卵巢，置于液氮中快速冷冻，转移至- 80 ℃保存。

1.4 酶联免疫吸附试验(enzyme-linked immunosor-bent assay，ELISA)测定血浆激素浓度

血浆E2、FSH和LH浓度ELISA检测试剂盒均购自上海西唐生物科技有限公司，试剂盒货号分别为F6010、F6006和F6005。AMH检测试剂盒购自中国无锡Develop公司，试剂盒货号为DL-AMH-Ra。具体操作步骤按照试剂盒说明书进行。根据各试剂盒标准曲线，结合各样本的光密度值，分别计算E2、FSH、LH和AMH的血清浓度。

1.5 蛋白质印迹法检测PPAR-α、PPAR-δ和PPAR-γ蛋白表达

剪取卵巢50~60 mg，液氮冷冻后研碎，加入裂解液(每10 mg组织加入100 mL裂解液)，静置20 min，12 000×g离心20 min，取上清。采用BCA法测定蛋白浓度，加入4×上样缓冲液，100 ℃变性10 min。每个样本取30 μg总蛋白进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，转膜，5%脱脂奶粉溶液封闭2 h。用Tris盐酸盐缓冲液+ Tween(TBST)洗膜3次，孵育PPAR-α、PPAR-δ、PPAR-γ和内参照α-tubulin一抗(均购自英国Abcam公司，稀释比例均为1 : 1 000)，4 ℃过夜。TBST洗膜3次后，室温孵育二抗2 h，TBST洗膜3次后进行化学发光显影。采用Gel-Pro Analyzer 1软件分析蛋白条带灰度值。

1.6 ELISA测定卵巢组织促炎细胞因子水平

组织白细胞介素(interleukin，IL)-1β、IL-6和肿瘤坏死因子α(tumor necrosis factor α，TNF-α)浓度ELISA检测试剂盒均购自上海西唐生物科技有限公司，试剂盒货号分别为F15810、F15870和F16960。同1.5项方法提取卵巢组织蛋白并测定蛋白浓度后，按照试剂盒说明书测定卵巢组织中IL-1β、IL-6和TNF-α的含量。根据各试剂盒标准曲线，结合各样本光密度值，分别计算IL-1β、IL-6和TNF-α的含量。

1.7 统计学处理

采用GraphPad Prism 6.0软件进行数据处理。数据均以x±s表示，2组间比较采用独立样本t检验；4个亚组间比较采用双因素方差分析，多重比较采用Tukey检验。检验水准(α)为0.05。

2 结果 2.1 LPS诱导慢性低度炎症对大鼠卵巢功能的影响

如表 1所示，无论大鼠处于发情周期任何阶段，相比NS组大鼠，腹腔注射LPS大鼠血清中E2和AMH水平均降低，而FSH和LH水平均升高，差异均有统计学意义(P均＜0.05)。

表 1 NS组与LPS组发情周期不同阶段大鼠血清中E2、FSH、LH和AMH的浓度比较 Tab 1 Comparison of serum E2, FSH, LH and AMH levels in rats at different stages of estrous cycle between NS and LPS groups

x±s
Parameter	Estrous cycle phase	NS group	LPS group	t value	P value
E2 ρ_B/(pg·mL^-1)	Proestrus n = 5	87.8±5.1	61.3±4.3	8.9	0.000 1
	Estrus n = 5	28.0±3.3	18.1±2.5	5.3	0.000 7
	Metestrus n = 5	29.1±3.8	16.1±2.6	6.2	0.000 3
	Diestrus n = 5	29.8±3.8	17.9±1.4	6.6	0.000 2
FSH ρ_B/(ng·mL^-1)	Proestrus n = 5	18.4±1.3	20.4±0.6	3.1	0.014 7
	Estrus n = 5	11.1±0.8	14.9±0.5	8.9	0.000 1
	Metestrus n = 5	8.0±0.8	10.9±0.8	5.9	0.000 4
	Diestrus n = 5	8.3±0.5	14.1±0.7	15.6	0.000 1
LH ρ_B/(ng·mL^-1)	Proestrus n = 5	11.7±0.8	14.6±0.6	6.2	0.000 3
	Estrus n = 5	10.5±0.6	14.1±1.1	6.3	0.000 2
	Metestrus n = 5	10.5±0.5	14.1±0.9	7.9	0.000 1
	Diestrus n = 5	10.8±1.0	13.0±1.1	3.2	0.012 8
AMH ρ_B/(ng·mL^-1)	Whole cycle n = 10	6.3±0.6	1.5±0.4	21.1	0.000 1
NS: Normal saline; LPS: Lipopolysaccharide; E2: Estradiol; FSH: Follicle stimulating hormone; LH: Luteinizing hormone; AMH: Anti-Müllerian hormone

2.2 LPS诱导慢性低度炎症对大鼠卵巢组织中PPAR表达的影响

如图 1所示，在发情周期不同阶段，卵巢组织中PPAR-α和PPAR-δ的蛋白表达在NS组和LPS组之间差异均无统计学意义(P均＞0.05)；而LPS组大鼠卵巢组织中PPAR-γ的蛋白表达水平均低于NS组，差异均有统计学意义(P均＜0.05)。

图 1 蛋白质印迹法检测2组大鼠发情周期不同阶段卵巢组织中PPAR-α、PPAR-δ和PPAR-γ蛋白表达 Fig 1 Comparison of PPAR-α, PPAR-δ and PPAR-γ protein expression in ovarian tissues of rats at different stages of estrous cycle in two groups detected by Western blotting A: Proestrus; B: Estrus; C: Metestrus; D: Diestrus. PPAR: Peroxisome proliferator-activated receptor; NS: Normal saline; LPS: Lipopolysaccharide. ^*P < 0.05 vs NS group. n = 5, x±s

2.3 罗格列酮对LPS诱导慢性低度炎症大鼠卵巢组织中促炎细胞因子的影响

如表 2所示，在发情周期不同阶段，相比NS + Veh亚组，LPS + Veh亚组大鼠的卵巢组织中促炎细胞因子IL-1β、IL-6和TNF-α的水平均升高(P均＜0.05)，而口服罗格列酮后即LPS + RSG亚组大鼠卵巢组织中促炎细胞因子IL-1β、IL-6和TNF-α水平均较LPS + Veh亚组降低(P均＜0.05)；但NS组大鼠口服罗格列酮(NS + RSG亚组)后，卵巢组织中上述促炎细胞因子的水平与溶剂对照干预大鼠相比无明显变化(P均＞0.05)。

表 2 罗格列酮对发情周期不同阶段LPS诱导慢性低度炎症大鼠卵巢组织中促炎细胞因子的影响 Tab 2 Effects of rosiglitazone on proinflammatory cytokines in ovarian tissues of rats with chronic low-grade inflammation induced by LPS at different stages of estrus cycle

n = 5, x±s
Parameter	Estrous cycle phase	NS + Veh	NS + RSG	LPS + Veh	LPS + RSG	F value	P value
IL-1β ρ_B/(pg·mL^-1)	Proestrus	107.8±2.3	109.6±3.3	168.7±8.8^*	134.7±4.1^△	146.3	0.000 1
	Estrus	106.8±3.7	101.0±1.7	163.9±9.1^*	131.5±2.8^△	152.8	0.000 1
	Metestrus	106.1±4.0	108.3±2.7	163.8±5.3^*	133.0±4.1^△	212.0	0.000 1
	Diestrus	101.9±7.5	105.1±3.5	166.5±6.7^*	126.9±5.1^△	127.3	0.000 1
IL-6 ρ_B/(ng·mL^-1)	Proestrus	340.3±8.2	336.4±29.9	625.6±35.1^*	446.2±62.1^△	60.8	0.000 1
	Estrus	365.4±27.2	341.0±33.7	591.2±52.7^*	428.2±28.2^△	46.6	0.000 1
	Metestrus	364.9±21.0	328.6±26.1	596.4±34.0^*	440.9±36.5^△	78.3	0.000 1
	Diestrus	335.5±12.6	356.9±25.7	601.2±26.8^*	421.3±24.3^△	136.9	0.000 1
TNF-α ρ_B/(ng·mL^-1)	Proestrus	31.6±4.6	30.2±4.5	64.3±9.6^*	42.2±2.6^△	35.5	0.000 1
	Estrus	32.1±2.6	31.3±4.2	58.9±5.7^*	41.4±3.2^△	49.1	0.000 1
	Metestrus	29.9±2.5	32.4±3.1	59.3±5.6^*	43.7±2.5^△	67.4	0.000 1
	Diestrus	32.1±2.2	31.9±2.3	57.7±4.7^*	43.4±2.5^△	78.2	0.000 1
NS + Veh: Intraperitoneal injection of normal saline + intragastric administration of normal saline subgroup; NS + RSG: Intraperitoneal injection of normal saline + intragastric administration of rosiglitazone subgroup; LPS + Veh: Intraperitoneal injection of lipopolysaccharide (LPS) + intragastric administration of normal saline subgroup; LPS + RSG: Intraperitoneal injection of LPS + intragastric administration of rosiglitazone subgroup. IL: Interleukin; TNF-α: Tumor necrosis factor α. ^*P＜0.05 vs NS + Veh subgroup; ^△P＜0.05 vs LPS + Veh subgroup

2.4 罗格列酮对LPS诱导慢性低度炎症大鼠卵巢功能的影响

如表 3所示，无论大鼠处于发情周期任何阶段，腹腔注射LPS后口服罗格列酮均可改善LPS诱导的大鼠血清中E2和AMH降低，同时抑制LPS诱导FSH和LH升高，与LPS + Veh亚组相比差异均有统计学意义(P均＜0.05)；但是腹腔注射生理盐水的大鼠，在发情周期的任何阶段血清中E2、FSH、LH和AMH水平均不受口服罗格列酮的影响(P均＞0.05)。

表 3 罗格列酮对发情周期不同阶段LPS诱导慢性低度炎症大鼠卵巢功能的影响 Tab 3 Effects of rosiglitazone on ovarian function in rats with LPS-induced chronic low-grade inflammation at different stages of estrous cycle

x±s
Parameter	Estrous cycle phase	NS + Veh	NS + RSG	LPS + Veh	LPS + RSG	F value	P value
E2 ρ_B/(pg·mL^-1)	Proestrus n = 5	89.4±3.4	90.2±3.4	62.9±5.6^*	75.6±4.9^△	31.6	0.000 1
	Estrus n = 5	27.3±2.3	27.7±2.1	17.6±1.8^*	21.5±1.4^△	31.8	0.000 1
	Metestrus n = 5	28.8±1.6	27.8±1.4	16.0±2.4^*	22.6±2.4^△	42.8	0.000 1
	Diestrus n = 5	29.5±2.9	32.6±5.4	17.0±1.5^*	23.0±1.6^△	22.4	0.000 1
FSH ρ_B/(ng·mL^-1)	Proestrus n = 5	18.3±0.9	18.5±1.1	24.8±1.4^*	19.9±0.9^△	37.4	0.000 1
	Estrus n = 5	10.5±1.2	10.3±0.9	16.6±1.1^*	13.8±0.8^△	42.8	0.000 1
	Metestrus n = 5	8.0±0.5	8.1±0.8	12.4±0.6^*	10.4±0.3^△	66.4	0.000 1
	Diestrus n = 5	8.3±0.4	8.0±0.3	14.3±0.5^*	11.2±0.8^△	157.5	0.000 1
LH ρ_B/(ng·mL^-1)	Proestrus n = 5	11.8±0.6	11.3±1.1	17.2±1.4^*	13.3±0.4^△	38.5	0.000 1
	Estrus n = 5	10.7±0.6	10.9±0.9	16.5±1.4^*	13.7±1.0^△	36.1	0.000 1
	Metestrus n = 5	10.7±0.8	10.7±0.6	16.3±0.7^*	13.9±0.5^△	98.4	0.000 1
	Diestrus n = 5	10.7±0.6	10.4±0.6	15.8±0.8^*	12.8±0.6^△	69.5	0.000 1
AMH ρ_B/(ng·mL^-1)	Whole cycle n = 40	6.2±0.6	5.9±0.4	1.1±0.3^*	4.6±0.2^△	1 346.0	0.000 1
NS + Veh: Intraperitoneal injection of normal saline + intragastric administration of normal saline subgroup; NS + RSG: Intraperitoneal injection of normal saline + intragastric administration of rosiglitazone subgroup; LPS + Veh: Intraperitoneal injection of lipopolysaccharide (LPS) + intragastric administration of normal saline subgroup; LPS + RSG: Intraperitoneal injection of LPS + intragastric administration of rosiglitazone subgroup. E2: Estradiol; FSH: Follicle stimulating hormone; LH: Luteinizing hormone; AMH: Anti-Müllerian hormone. ^*P＜0.05 vs NS + Veh subgroup; ^△P＜0.05 vs LPS + Veh subgroup

3 讨论

卵巢功能减退为卵巢早衰前期，常无特异性临床表现，其主要表现可能为月经周期紊乱、经量减少和不孕等^[17-18]。卵巢功能状态对于育龄期女性生殖健康尤为重要，但是导致卵巢功能异常的可能机制众多，如肥胖^[19]、产后宫内感染^[20]、接触化学治疗药物^[21]或组蛋白去乙酰化酶异常活化^[22]等，阐明卵巢功能异常的病理生理学机制对卵巢早衰的防治具有重要的临床意义。

全身低度炎症作为一种慢性疾病状态，其特征是机体对有害刺激的反应导致循环中炎性介质增多，包括经典的促炎分子如IL-1、IL-6、TNF-α和C-反应蛋白^[23]。研究表明慢性低度炎症状态与肥胖、胰岛素抵抗和心血管疾病的发生、发展密切相关^[24]。在临床研究中，慢性低度炎症的分子标志物C-反应蛋白水平升高可预测心肌梗死、脑卒中等心血管疾病^[25-26]。一项基于人群的队列研究发现男性不育可能与IL-6水平升高有关^[27]；多囊卵巢综合征患者促炎细胞因子尤其是IL-1过度活跃^[3]。以上这些结果表明慢性低度炎症与不孕不育症密切相关。本研究在LPS诱导的慢性低度炎症大鼠模型上，观察到慢性低度炎症大鼠血清中E2和AMH水平降低，同时FSH、LH、卵巢组织中促炎细胞因子水平升高提示慢性低度炎症可诱导卵巢炎症反应，减弱了卵巢产生雌激素的能力。卵巢合成雌激素受FSH和LH的调控，因此本研究的结果提示了慢性低度炎症对中枢神经系统尤其是下丘脑-腺垂体影响不大，慢性低度炎症引起的E2和AMH水平降低可促使机体负反馈调节FSH和LH能力减弱，导致FSH和LH水平升高。

为进一步探究慢性低度炎症导致卵巢雌激素产生能力下降的可能机制，本研究检测了卵巢组织中PPAR的表达情况，结果显示PPAR-γ在慢性低度炎症大鼠的卵巢组织中表达降低，而口服罗格列酮特异性激活PPAR-γ后可降低慢性低度炎症大鼠卵巢组织中促炎细胞因子的水平，并改善了卵巢组织产生雌激素的能力和卵巢储备功能。除此之外，本研究初步探究了PPAR-γ激动剂罗格列酮改善慢性低度炎症诱导的卵巢功能异常的效应，为卵巢功能的保护提供了新的理论依据。

雌性大鼠发情周期为4~5 d，主要分为发情前期、发情期、发情后期和发情间期4个阶段，每个阶段阴道黏膜细胞、性腺类固醇激素和促性腺激素浓度会发生周期性变化，为确定雌激素水平的变化是由慢性低度炎症所致还是口服罗格列酮治疗所致，实验中则有必须排除发情周期所引起雌激素水平波动的干扰。因此，本研究的一大创新点在于检测了处于发情周期不同阶段的大鼠血清中激素水平、卵巢组织中PPAR表达和促炎细胞因子的变化，特别是当NS组大鼠与LPS组大鼠进行比较时，在观察2组间上述指标的差异受慢性低度炎症的影响时可以有效地排除发情周期引起的雌激素水平波动对研究结果的干扰。

本研究结果表明慢性低度炎症引起的卵巢功能异常与PPAR-γ活性降低有关，罗格列酮可改善慢性低度炎症诱导的卵巢功能异常，为阐明慢性低度炎症诱导卵巢功能异常的分子机制及探究卵巢功能保护提供了理论依据。但本研究仍存在不足之处：(1)因慢性低度炎症大鼠卵巢组织中PPAR-α和PPAR-δ的蛋白表达与NS组相比没有明显变化，仅PPAR-γ表达降低，故本研究仅针对PPAR-γ进行了干预，而缺乏对PPAR-α和PPAR-δ的处理，无法确定PPAR-γ在慢性低度炎症诱导卵巢功能异常中的特异性。(2)全身慢性低度炎症是通过腹腔注射LPS实现的，而LPS主要是通过与Toll样受体4特异性结合发挥促炎效应，产生大量的促炎细胞因子^[28]，通过腹腔注射LPS诱导慢性低度炎症引起雌激素产生减少的确切分子机制仍未明确，需要进一步研究以明确LPS诱导的慢性低度炎症引起雌激素产生减少是由LPS直接作用于Toll样受体4介导，还是由于LPS诱导促炎细胞因子，从而降低卵巢颗粒细胞芳香化酶的活性，进而减少雌激素产生。

基于本研究结果和分析，慢性低度炎症引起的卵巢功能异常与PPAR-γ表达降低有关，并且罗格列酮可通过减轻卵巢炎症反应改善慢性低度炎症诱导的卵巢功能异常。因此，PPAR-γ激动剂罗格列酮对于卵巢功能具有直接或间接的保护作用，但是其对卵巢功能异常导致的女性不孕的疗效仍需进一步证实和评估。

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第二军医大学学报 2019, Vol. 40 Issue (12): 1310-1316	PDF