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2024 Volume 46 Issue 9
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ZHANG Lifang, JIANG Shengming, MA Qi, et al. Effects of Pulsatilla Decoction on the Barrier Function of Ileal Mucosa in Rats with Damp-Heat Diarrhoea[J]. Journal of Southwest University Natural Science Edition, 2024, 46(9): 85-96. doi: 10.13718/j.cnki.xdzk.2024.09.008
Citation: ZHANG Lifang, JIANG Shengming, MA Qi, et al. Effects of Pulsatilla Decoction on the Barrier Function of Ileal Mucosa in Rats with Damp-Heat Diarrhoea[J]. Journal of Southwest University Natural Science Edition, 2024, 46(9): 85-96. doi: 10.13718/j.cnki.xdzk.2024.09.008
shu

Effects of Pulsatilla Decoction on the Barrier Function of Ileal Mucosa in Rats with Damp-Heat Diarrhoea

  • College of Veterinary Medicine, Southwest University, Rongchang Chongqing 402460, China

More Information
  • Corresponding author: MA Qi ; 
  • Received Date: 19/08/2023
    Available Online: 20/09/2024

  • MSC: S853.6

  • To investigate the effect of Pulsatilla Decoction (PD) on the mucosal barrier function of ileum of rats with Damp-heat Diarrhea (DHD).Forty-eight male SD rats were selected and randomly divided into blank group (NC), model group (Model), self-healing group (SH), pulsatilla high dose group (PD-H), pulsatilla medium dose group (PD-M), and pulsatilla low dose group (PD-L).The rat DHD model was induced by the complex factors of "high sugar, high fat, high heat, high humidity and enterotoxigenic E. coli".After the model was successfully established, rats in the PD group were treated with 0.752 g/mL (PD-H), 0.376 g/mL (PD-M), and 0.188 g/mL (PD-L) by gavage, respectively, while the rats in the SH and NC groups were garaged with an equal amount of saline once a day for 5 d.During the test period, the changes of body weight and food intake of the rats were recorded; at the end of the test period, blood was collected for the detection of hematological indexes and determination of plasma inflammatory cytokine content.Heart, liver, spleen, lungs and kidneys were collected and weighed to calculate the organ indexes.Ileal tissues were collected for pathological and histological observation and detection of inflammatory reactions, and expression levels of the genes related to mucosal barrier and tryptophan pathway.The results showed that compared with the NC group, rats in the Model group had lower feed intake, lower growth rate of body mass, significantly higher heart, liver, spleen, lung and kidney organ indexes (p < 0.05), significantly lower Lymph, Mon and MCV (p < 0.05), significantly higher Gran, Lymph%, RBC and PLT (p < 0.05), significantly higher serum IL-1β and IL-6 (p < 0.05), and disorganized ileal villi with significant thickening of the lamina propria (p < 0.05).Some of the crypt structures were disappeared, the number of cup cells was decreased, the striatal rim formed by microvilli was diffusely missing.There was erythrocyte infiltration in the celiac duct, the height of the intestinal villi/crypt depth was significantly reduced (p < 0.05).The mRNA levels of ileal IL-1β, IL-6 and IFN-γ, TNF-α, and IL-17 were significantly increased (p < 0.05), the mRNA levels of IL-4 and ZO-1 were significantly reduced (p < 0.05), and the mRNA levels of TPH1, KMO, IDO-1, MUC1, MUC2 were significantly increased (p < 0.05).Compared with the Model group, Lymph and MCV were significantly increased in the PD-H group (p < 0.05), Lymph, Mon and PLT were significantly decreased in the PD-M group (p < 0.05), HGB was significantly decreased and MCV was significantly increased in the PD-L group (p < 0.05), liver index was significantly decreased in the PD-M (p < 0.05).The rats in PD-H, PD-M, PD-L had significantly lower spleen indices (p < 0.05), the rats in PD-H and PD-L had significantly lower lung and kidney indices (p < 0.05), intestinal villi of the rats in PD-H and PD-M were intact and neatly arranged, with varying thicknesses of the lamina propria, obvious crypts, increased or neatly arranged cup cells, intact striated rims, and erythrocyte infiltration in the widening of celiac ducts.The ratio of villus height/crypt depth (V/C) in the PD-H group was significantly increased (p < 0.05).The levels of plasma IL-1β and IL-6 were significantly reduced (p < 0.05), the mRNA levels of TPH1 and KMO in the ileum of rats in the PD-H, PD-M, and PD-L groups were significantly reduced (p < 0.05), the mRNA levels of MUC1 in the PD-H and PD-M groups were significantly reduced (p < 0.05), the mRNA levels of MUC2 in the PD-H, PD-M, and PD-L groups were extremely significantly reduced (p < 0.01), and the mRNA levels of ZO-1 in the PD-H and PD-M groups were extremely significantly increased (p < 0.01).The results showed that pulsatilla decoction could improve the inflammatory response, regulate tryptophan metabolism, alleviate the mucosal barrier damage, and maintain the normal function of the ileum.

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Effects of Pulsatilla Decoction on the Barrier Function of Ileal Mucosa in Rats with Damp-Heat Diarrhoea

    Corresponding author: MA Qi ; 
  • Received Date: 19/08/2023
  • Available Online: 20/09/2024

Abstract: 

To investigate the effect of Pulsatilla Decoction (PD) on the mucosal barrier function of ileum of rats with Damp-heat Diarrhea (DHD).Forty-eight male SD rats were selected and randomly divided into blank group (NC), model group (Model), self-healing group (SH), pulsatilla high dose group (PD-H), pulsatilla medium dose group (PD-M), and pulsatilla low dose group (PD-L).The rat DHD model was induced by the complex factors of "high sugar, high fat, high heat, high humidity and enterotoxigenic E. coli".After the model was successfully established, rats in the PD group were treated with 0.752 g/mL (PD-H), 0.376 g/mL (PD-M), and 0.188 g/mL (PD-L) by gavage, respectively, while the rats in the SH and NC groups were garaged with an equal amount of saline once a day for 5 d.During the test period, the changes of body weight and food intake of the rats were recorded; at the end of the test period, blood was collected for the detection of hematological indexes and determination of plasma inflammatory cytokine content.Heart, liver, spleen, lungs and kidneys were collected and weighed to calculate the organ indexes.Ileal tissues were collected for pathological and histological observation and detection of inflammatory reactions, and expression levels of the genes related to mucosal barrier and tryptophan pathway.The results showed that compared with the NC group, rats in the Model group had lower feed intake, lower growth rate of body mass, significantly higher heart, liver, spleen, lung and kidney organ indexes (p < 0.05), significantly lower Lymph, Mon and MCV (p < 0.05), significantly higher Gran, Lymph%, RBC and PLT (p < 0.05), significantly higher serum IL-1β and IL-6 (p < 0.05), and disorganized ileal villi with significant thickening of the lamina propria (p < 0.05).Some of the crypt structures were disappeared, the number of cup cells was decreased, the striatal rim formed by microvilli was diffusely missing.There was erythrocyte infiltration in the celiac duct, the height of the intestinal villi/crypt depth was significantly reduced (p < 0.05).The mRNA levels of ileal IL-1β, IL-6 and IFN-γ, TNF-α, and IL-17 were significantly increased (p < 0.05), the mRNA levels of IL-4 and ZO-1 were significantly reduced (p < 0.05), and the mRNA levels of TPH1, KMO, IDO-1, MUC1, MUC2 were significantly increased (p < 0.05).Compared with the Model group, Lymph and MCV were significantly increased in the PD-H group (p < 0.05), Lymph, Mon and PLT were significantly decreased in the PD-M group (p < 0.05), HGB was significantly decreased and MCV was significantly increased in the PD-L group (p < 0.05), liver index was significantly decreased in the PD-M (p < 0.05).The rats in PD-H, PD-M, PD-L had significantly lower spleen indices (p < 0.05), the rats in PD-H and PD-L had significantly lower lung and kidney indices (p < 0.05), intestinal villi of the rats in PD-H and PD-M were intact and neatly arranged, with varying thicknesses of the lamina propria, obvious crypts, increased or neatly arranged cup cells, intact striated rims, and erythrocyte infiltration in the widening of celiac ducts.The ratio of villus height/crypt depth (V/C) in the PD-H group was significantly increased (p < 0.05).The levels of plasma IL-1β and IL-6 were significantly reduced (p < 0.05), the mRNA levels of TPH1 and KMO in the ileum of rats in the PD-H, PD-M, and PD-L groups were significantly reduced (p < 0.05), the mRNA levels of MUC1 in the PD-H and PD-M groups were significantly reduced (p < 0.05), the mRNA levels of MUC2 in the PD-H, PD-M, and PD-L groups were extremely significantly reduced (p < 0.01), and the mRNA levels of ZO-1 in the PD-H and PD-M groups were extremely significantly increased (p < 0.01).The results showed that pulsatilla decoction could improve the inflammatory response, regulate tryptophan metabolism, alleviate the mucosal barrier damage, and maintain the normal function of the ileum.

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  • 湿热泄泻(Damp-heat Diarrhea，DHD)又称泄泻·湿热蕴结证，指机体在外源致病因子、饮食不节/洁等因素影响下湿热之邪蕴结于肠内，以热证、湿证为主的泄泻证候.相关研究显示，DHD大鼠回肠杯状细胞的密度极显著降低、潘氏细胞的密度极显著升高[1]；DHD仔猪回肠中大肠杆菌数量极显著上升，乳酸杆菌数量显著降低，乳酸杆菌与大肠杆菌比值极显著下降，且回肠IL-2 mRNA表达量显著上升，IL-4，IL-5 mRNA表达量显著降低[2]；DHD大鼠回肠绒毛上皮脱落、黏膜固有层出血[3]；DHD仔猪回肠Notch-1，Hes-1 mRNA的表达量显著升高、Hath-1 mRNA的表达量显著降低[4].

    白头翁汤(Pulsatilla Decoction，PD)出自《伤寒杂病论》，是由白头翁、黄连、黄柏、秦皮组成的传统中药方剂，具有清热解毒、凉血止痢的功效，为治疗热毒痢疾的原始处方.方中以白头翁为君，清热解毒，凉血止痢；臣以黄连之苦寒，清热解毒，燥湿厚肠；黄柏泻下焦湿热，共奏燥湿止痢之效；秦皮苦寒性涩，收敛作用强，用以止血.四药并用，为热毒血痢之良方，临床上常用于治疗阿米巴痢疾、细菌性痢疾等病证.近年来，PD治疗肠道病证的相关研究发现，PD通过显著降低血清中IL-2，IFN-γ，DAO，HIF-1α及CD8+水平且显著提高IL-4，IL-10，CD4+以及CD4+/CD8+水平，来调节Th1/Th2细胞平衡和激活免疫机制[5]；PD可显著降低小鼠血清中IL-6和TNF-α水平，显著降低结肠组织中p-mTOR/mTOR，p-P70S6K/P70S6K，p-STAT3/STAT3，COX-2 mRNA的表达量，从而预防小鼠溃疡性结肠炎[6].PD可抑制TNF-α mRNA的表达量从而抑制溃疡性结肠炎的炎症反应，促进肠黏膜愈合[7]；PD可抑制TLR4/NF-κB信号通路，下调P-选择素，MPO，MIF，TXB2的水平，促进肠道黏膜的修复，减轻溃疡性结肠炎大鼠结肠炎症反应[8-9].

    本课题组前期研究发现，PD对DHD有良好的治疗效果，可以缓解DHD肠道组织损伤[9].为进一步探究PD对DHD大鼠回肠黏膜屏障功能及色氨酸代谢通路的影响，本试验建立DHD大鼠模型，并以不同剂量PD进行灌胃治疗，分析大鼠回肠组织形态变化、回肠肠道黏膜屏障损伤、肠道炎症反应及色氨酸代谢通路之间的关联，以期为探索PD介导改善DHD的潜在机制提供新见解.

1.   材料与方法

    1.1.   材料

    1.1.1.   试验动物

  • 48只SPF级雄性SD大鼠，7周龄，体质量180~200 g，购自湖南斯莱克景达实验动物有限公司[SCXK(湘)2019-0004]，饲养于西南大学动物医学院实验室[SYXK(渝)2019-0003]，造模前适应性饲养7 d，自由采食及饮水，环境温度为20~24 ℃.所有操作均符合西南大学动物实验伦理学要求(审批号：IACUC-20210507-06).

    • 1.1.2.   主要试剂与仪器

  • 大肠杆菌(南京农业大学分离鉴定的HE株[10])由南京农业大学馈赠；蜂蜜(GB14963)：江西华茂保健品开发有限公司；猪油：重庆市荣昌区水口寺菜市场；56度红星二锅头(GB/T10781.2)：北京红星股份有限公司；苏木素伊红染色液(20220610)：湖南比克曼生物科技有限公司；Rat IL-1β ELISA KIT(YJ003075)及Rat IL-6 ELISA KIT(YJ064292)：上海源桔生物科技中心；TRIzol试剂：Beyotime，上海；PrimeScriptTM RT Master Mix(RR036A)及TB Green © Premix Ex TaqTM Ⅱ(RR820A)：宝日医生物技术(北京)有限公司；兽用全自动血液细胞分析仪(BC-2600Vet)：深圳迈瑞生物医疗电子股份有限公司；轮式切片机(KD1508A)：浙江金华科迪仪器设备有限公司；全自动酶标仪(A-5082)：Tecan Austria GmbH；实时荧光定量PCR仪(Archimed X6)：杭州鲲鹏基因科技有限责任公司.

    • 1.2.   方法

    1.2.1.   白头翁汤的制备

  • 白头翁、秦皮、黄连、黄柏购置于西城大药房(中国重庆荣昌)药材公司，经过西南大学动物医学院曹立亭副教授鉴定，药材信息如表 1所示.根据2020年版《中华人民共和国兽药典》关于白头翁散的配比称取白头翁60 g、秦皮45 g、黄连30 g、黄柏60 g，加10倍水浸泡30 min，大火煮沸后转小火煎煮40 min，过滤收集滤液后再加8倍量水继续煎煮，如此重复2次，合并3次滤液，浓缩至0.188 g/mL作为PD低浓度、0.376 g/mL作为PD中浓度、0.752 g/mL作为PD高浓度.

    • 1.2.2.   动物分组及处理

  • 试验前1 d，称量每只SD大鼠体质量，用分层随机法将48只大鼠随机分为空白组(NC)、模型组(Model)、自愈组(SH)、白头翁汤高剂量组(PD-H)、白头翁汤中剂量组(PD-M)、白头翁汤低剂量组(PD-L)，每组8只大鼠，平均体质量为(254±3)g.除NC外，分3个阶段建立DHD模型，分别为高糖高脂、高热高湿及攻毒阶段，全程给予30%蜂蜜水.高糖高脂阶段为1~10 d，第1，3，5，7，9 d给予每只大鼠4 mL猪油，自由采食和饮用蜂蜜水，第2，4，6，8，10 d禁食不禁水；高热高湿阶段为11~15 d，此阶段给予每只大鼠2 mL红星二锅头，自由采食饮水，每日需在自建高温湿度棚[温度(34±1) ℃，湿度(94±1) %]放置8 h；攻毒阶段为16~18 d，Model、SH、PD-H、PD-M、PD-L大鼠于第16 d及17 d注射浓度为3.96×1011 CFU/mL大肠杆菌0.2 mL，此阶段均自由采食及饮水.NC饲喂方式与适应性饲养方法相同.治疗阶段为19~23 d，造模成功后，PD-H、PD-M、PD-L大鼠分别灌胃不同剂量PD并于第19 d处死Model组大鼠.于造模前后每日定时记录各组大鼠体质量、采食量，各组大鼠实验处理情况如表 2所示.

    • 1.2.3.   样品采集

  • 试验结束后，称各组大鼠体质量并将各组大鼠处死及时称质量和记录大鼠心、肝、脾、肺、肾的质量，根据各脏器质量/体质量得到大鼠各脏器指数.采集各组大鼠回肠组织取适宜长度固定于4%中性甲醛溶液，另取一部分回肠组织保存于-80 ℃冰箱备用.

    • 1.2.4.   血液指标检测

  • 采集各组大鼠20 μL尾尖血，加入稀释液轻轻吹打混匀，于西南大学动物医院对大鼠血常规指标进行检测.腹主动脉采集血液并分离血浆，按照ELISA试剂盒说明书要求测定大鼠血浆IL-6，IL-1β.

    • 1.2.5.   回肠组织形态学观察

  • 取固定的各组大鼠回肠组织，冲水后经75%~100%乙醇梯度脱水并透明，浸蜡1.5 h，重复2次.包埋后切片、展片，烘片后使用二甲苯浸泡脱蜡，重复2次再经100%~75%乙醇梯度复水，苏木素伊红染色后75%~100%乙醇梯度脱水，二甲苯透明后即可用中性树胶封片以备回肠组织形态学观察，并测定回肠绒毛高度和隐窝深度，计算绒毛高度和隐窝深度的比值.

    • 1.2.6.   回肠炎症反应、色氨酸代谢及肠道黏膜屏障mRNA表达水平测定

  • 取-80 ℃储存的各组大鼠回肠组织用TRIzol试剂提取总RNA，利用PrimeScriptTM RT Master Mix试剂盒说明书(含gDNase)反转录获得cDNA，存于-20 ℃备用.使用QuantStudioTM 7 Flex实时荧光定量PCR系统和SuperReal PreMix Plus(SYBR Green)进行qPCR. RT-qPCR反应体系为TB Green ©Premix Ex TaqTMⅡ(2×)5 μL，上、下游引物各0.4 μL，ROX Reference Dye (50×)0.2 μL，无酶无菌水3 μL，DNA模板1 μL.以GAPDH作为内参基因.反应条件为95 ℃预变性30 s；95 ℃ 5 s变性，60 ℃ 34 s退火，反应40个循环60 ℃ 1 min延伸，95 ℃ 15 s熔解.数据分析采用2-ΔΔCt方法.使用的引物序列如表 3所示.

    • 1.2.7.   统计学分析

  • 结果以平均值±标准差表示，用IBM SPSS 26.0软件分析试验数据，并使用单因素方差分析(ANOVA)组间差异，然后进行LSD事后检验，p＜0.05表示差异有统计学意义.用Prism Grappad 9.0作图.

2.   结果与分析

    2.1.   PD对DHD大鼠体质量和采食量的影响

  • 如图 1a所示，试验中高糖高脂、高热高湿、攻毒及治疗阶段大鼠采食量起伏波动：高糖高脂阶段大鼠采食量初上升后平稳，与NC组相比，Model组大鼠采食量明显降低，SH组、PD-H组、PD-M组、PD-L组大鼠与Model组相比，差异无统计学意义；高热高湿阶段大鼠采食量先急剧下降后逐渐平稳，与NC组相比，Model组大鼠采食量明显下降，SH组、PD-H组、PD-M组、PD-L组与Model组相比，差异无统计学意义；攻毒阶段，与NC组相比，Model组大鼠采食量明显降低，SH组、PD-H组、PD-M组、PD-L组与Model组相比，差异无统计学意义；治疗阶段，与NC组相比，SH组大鼠采食量明显降低，与SH组相比，PD-H组、PD-M组、PD-L组大鼠采食量明显上升，且PD-M组大鼠采食量高于PD-H组和PD-L组.

    如图 1b所示，试验中高糖高脂、高热高湿、攻毒及治疗阶段大鼠体质量增长率各不相同：Model组、SH组、PD-H组、PD-M组和PD-L组大鼠高糖高脂阶段体质量变化率分别为6.7%，8.9%，9.0%，5.0%，7.1%，低于NC组的45.6%；高热高湿阶段Model组、SH组、PD-H组、PD-M组、PD-L组大鼠体质量变化率分别为22.2%，28.6%，26.2%，23.8%，26.4%，低于NC组的68.4%；攻毒阶段Model组、SH组、PD-H组、PD-M组、PD-L组大鼠体质量变化率分别为20.0%，21.5%，23.8%，17.8%，20.8%，均低于NC组的76.8%；治疗阶段，PD-H组、PD-M组、PD-L组大鼠体质量变化率均高于SH组大鼠，且4组均低于NC组.

    • 2.2.   PD对DHD大鼠血常规指标的影响

  • 如表 4所示，造模成功后，Model组大鼠的淋巴细胞数(Lymph)、单核细胞数(Mon)、中性粒细胞绝对值(Gran)、淋巴细胞百分比(Lymph%)、红细胞数(RBC)、平均红细胞体积(MCV)、血小板数(PLT)都发生了显著变化：与NC组相比，Model组大鼠Lymph，Mon，MCV显著降低(p＜0.05)，Gran，Lymph%，RBC，PLT显著升高(p＜0.05)，而SH组大鼠白细胞总数(WBC)、血红蛋白数(HGB)均显著降低(p＜0.05)，即WBC，HGB显著改变发生于DHD大鼠自愈恢复期.与Model相比，PD-H组大鼠Lymph，MCV显著升高(p＜0.05)；PD-M组大鼠Lymph，Mon，PLT均显著降低(p＜0.05)，而HGB，MCV显著升高(p＜0.05)；PD-L组大鼠HGB显著降低、MCV显著升高(p＜0.05).

    • 2.3.   PD对DHD大鼠脏器指数的影响

  • 如表 5所示，造模后，Model组大鼠的心脏、肝脏、脾脏、肺脏、肾脏指数与NC组比较均显著上升(p＜0.05).与Model组相比，PD-H组、PD-M组、PD-L组大鼠情况各异：PD-H组、PD-L组大鼠心脏指数有降低趋势，但差异无统计学意义；PD-M组大鼠肝脏指数显著降低(p＜0.05)，PD-H组、PD-L组大鼠有所降低，但差异无统计学意义，PD-H组、PD-M组、PD-L组大鼠脾脏指数均显著降低(p＜0.05)，PD-H组、PD-L组大鼠肺脏、肾脏指数显著降低(p＜0.05)，PD-M组大鼠有降低趋势，但差异无统计学意义；SH组大鼠肝脏、脾脏、肺脏指数均显著降低(p＜0.05).

    • 2.4.   PD对DHD大鼠回肠组织形态的影响

  • 图 2a显示，NC组大鼠回肠在低倍镜下形态结构完整，肠绒毛致密，肠绒毛排列整齐无断裂，固有层连接紧密无增厚；高倍镜下绒毛轮廓清晰，隐窝结构清晰完整，微绒毛形成的纹状缘清晰完整，杯状细胞排列整齐，乳糜管结构清晰无异常.图 2b显示，Model组大鼠回肠低倍镜下结构完整，肠绒毛过于密集，肠绒毛排列较杂乱，固有层明显增厚；高倍镜下隐窝结构有消失，杯状细胞数量减少，微绒毛形成的纹状缘弥漫性缺失，乳糜管内有红细胞浸润，说明有出血.图 2c显示，SH组大鼠回肠低倍镜下结构紊乱，肠绒毛形状各异、排列紊乱，固有层厚薄不一；高倍镜下隐窝结构有消失，杯状细胞排列紊乱，纹状缘弥漫性缺失，乳糜管增宽内有红细胞浸润及炎性细胞，说明有出血和炎症.图 2d显示，PD-H组大鼠回肠低倍镜下结构完整，肠绒毛形状完整、排列整齐，固有层厚薄不一；高倍镜下隐窝结构明显，杯状细胞有增多、排列整齐，乳糜管增宽内有红细胞浸润，说明有出血.图 2e显示，PD-M组大鼠回肠低倍镜下结构完整，肠绒毛形状各异、排列紧密而紊乱，固有层厚薄不一；高倍镜下隐窝有消失，杯状细胞有增多、排列紊乱，乳糜管增宽内有红细胞浸润，说明有出血.图 2f显示，PD-L组大鼠回肠低倍镜下结构完整，肠绒毛形状各异、排列疏松而紊乱，固有层变薄；高倍镜下隐窝变短，杯状细胞排列紊乱，乳糜管增宽内有红细胞浸润，说明有出血.

    测定各组大鼠回肠绒毛高度、隐窝深度，计算绒毛高度与隐窝深度的比值[11]，结果如图 3a所示.与NC组比较，Model组的变化并不显著，但SH组大鼠回肠绒毛高度显著降低(p＜0.05)；与SH组比较，PD-H组、PD-M组回肠绒毛高度显著升高(p＜0.05).如图 3b所示，与NC组比较，Model组大鼠回肠隐窝深度显著升高(p＜0.05)；与Model组比较，PD-H组、PD-M组回肠隐窝深度显著降低(p＜0.05).如图 3c所示，与NC组比较，Model组大鼠回肠绒毛高度/隐窝深度的值显著降低(p＜0.05)；与Model组比较，PD-H组大鼠回肠绒毛高度/隐窝深度的值显著升高(p＜0.05).

    • 2.5.   PD对DHD大鼠炎症细胞因子血浆质量浓度及mRNA表达量的影响

  • 如图 4a，e所示，与NC组比较，Model组大鼠血浆IL-1β质量浓度与回肠组织IL-1β mRNA表达量极显著升高(p＜0.01)；与Model组比较，PD-H组、PD-M组大鼠显著降低(p＜0.05)，PD-L组大鼠降低但不显著；SH组大鼠IL-1β质量浓度、mRNA表达量均未恢复.如图 4b，f所示，与NC组比较，Model组大鼠血浆IL-6质量浓度与回肠组织IL-6 mRNA表达量均显著升高(p＜0.05)；与Model组比较，PD-H组大鼠血浆IL-6质量浓度显著降低(p＜0.05)，PD-H组、PD-M组、PD-L组大鼠回肠组织IL-6 mRNA表达量均显著降低(p＜0.05).

    如图 4c所示，与NC组比较，Model组大鼠回肠组织IL-4 mRNA表达量显著降低(p＜0.05)；与Model组比较，PD-H组、PD-M组、PD-L组大鼠回肠IL-4 mRNA表达量无显著差异.如图 4g所示，与NC组比较，Model组大鼠回肠IL-17 mRNA表达量显著升高(p＜0.05)；与Model组比较，PD-H组大鼠回肠IL-17 mRNA表达量极显著降低(p＜0.01)、PD-M组显著降低(p＜0.05).如图 4d所示，与NC组比较，Model组大鼠回肠组织TNF-α mRNA表达量显著升高(p＜0.05)；与Model组比较，PD-H组、PD-L组大鼠回肠组织TNF-α mRNA表达量显著降低(p＜0.05).如图 4h所示，与NC组比较，Model组大鼠回肠组织IFN-γ mRNA表达量显著升高(p＜0.05)；与Model组比较，PD-H组、PD-M组、PD-L组大鼠回肠组织IFN-γ mRNA表达量显著降低(p＜0.05).

    • 2.6.   PD对DHD大鼠回肠色氨酸代谢相关基因表达量的影响

  • 如图 5所示，与NC组比较，除KYNU外，Model组大鼠回肠组织TPH1，KMO，IDO-1 mRNA表达量均显著升高(p＜0.05).与Model组比较，PD-H组、PD-M组、PD-L组大鼠回肠组织TPH1，KMO mRNA水平均显著降低(p＜0.05)，PD-H组的KYNU mRNA水平显著降低，PD-H组、PD-M组大鼠回肠组织IDO-1 mRNA水平显著降低(p＜0.05).

    • 2.7.   PD对DHD大鼠回肠肠道黏膜屏障相关基因mRNA表达量的影响

  • 如图 6所示，与NC组比较，Model组大鼠回肠组织的MUC1，MUC2 mRNA表达水平显著升高(p＜0.05)，ZO-1 mRNA表达水平显著降低(p＜0.05).与Model组比较，PD-H组、PD-M组、PD-L组治疗后大鼠的MUC1，MUC2 mRNA水平均降低，其中，PD-H组、PD-M组显著降低MUC1的mRNA表达水平(p＜0.05)；PD-H组、PD-M组、PD-L组极显著降低MUC2的mRNA表达水平(p＜0.01)；PD-H组极显著升高ZO-1的mRNA表达水平(p＜0.01)，PD-M组显著升高ZO-1的mRNA表达水平(p＜0.05).

3.   讨论与结论

    3.1.   讨论

  • 本试验各组大鼠血常规指标的差异说明DHD大鼠处于炎症阶段，Model组、SH组大鼠Lymph，Mon显著降低，而经不同剂量的PD治疗后大鼠的Lymph，Mon显著回升或有所回升，说明大鼠处于DHD阶段机体血液中淋巴细胞、单核细胞显著降低，这无疑增加机体继发感染的风险，降低了机体的免疫力，而不同剂量的PD均不同程度地阻止了淋巴细胞、单核细胞数目的减少，有利于维持机体的抗感染环境，并能有效阻止炎性细胞减少.Model组大鼠Gran，Lymph%显著上升而经PD治疗后有所减少，说明DHD大鼠机体内有大量致病因子，Gran也被称为“小吞噬细胞”，一方面Gran的增多有利于吞噬病原、衰老损伤的细胞及细胞因子，有利于增强细胞免疫反应[12]，但另一方面Gran过多也不利于机体炎性稳态，可能会损伤其他健康细胞.Model组大鼠RBC显著上升、MCV显著降低，结合DHD大鼠出现的腹泻、血便等临床症状说明DHD大鼠腹泻情况较为严重，机体存在脱水现象，而经PD治疗后RBC回调或显著阻止MCV降低，说明PD可缓解DHD大鼠的腹泻脱水症状.Model组大鼠PLT显著升高，结合回肠组织病理切片结果可得DHD大鼠回肠处有出血，需血小板发挥止血作用，因此，Model组大鼠血小板数量急剧上升，PD可显著缓解DHD大鼠的出血症状，阻止血小板的急剧增多，这有利于阻止血栓的形成从而保护DHD大鼠回肠.

    DHD大鼠的IL-1β，IL-6 mRNA表达量显著升高，而经PD治疗后DHD大鼠回肠组织IL-1β，IL-6 mRNA表达量显著降低.Kaminsky等[13]研究发现IL-1β通过丝裂原活化蛋白激酶(MAPKs)信号转导途径和其他转录因子诱导增加肠道通透性.IL-6与其他炎症细胞因子相互作用，引发炎症反应[14]，Guo等[15]研究发现IL-6介导的Jak/STAT3途径可能通过其下游PI3激酶/Akt信号肽驱动杯状细胞分化，IL-6是损伤后组织修复所必需的，能促进隐窝中潘氏细胞的增殖从而诱导肠上皮增殖，IL-6有助于通过经典信号传导和反式信号通路维持肠道稳态.本研究中，DHD大鼠回肠组织IL-1β，IL-6的mRNA表达量显著升高，可能会增加回肠通透性，易引起炎性风暴，会增加对机体的损伤[16].PD治疗后，DHD大鼠血浆IL-1β，IL-6质量浓度和回肠组织mRNA表达量回调，表明PD可能通过保护回肠的通透性、降低炎症、促进回肠上皮增生来保护回肠，减轻回肠损伤.

    本研究中，DHD大鼠的IL-4表达量显著降低，IL-17，IFN-γ，TNF-α mRNA表达量显著升高.研究表明，回肠组织IL-4 mRNA的表达量可反映出回肠黏膜炎症的变化趋势，IL-4不仅能促进淋巴细胞分化、使其增殖能力增强，还可促进体液免疫增强，刺激B细胞、嗜酸性粒细胞、嗜碱性粒细胞、肥大细胞的活化[17]，而且IL-4具有一定的免疫抑制作用.Lee等[18]研究发现，IL-17缺乏会降低DSS诱导的肠黏膜损伤期间维持屏障功能的能力，IFN-γ，TNF-α影响上皮通透性[19]，增加肠道通透性，从而进一步暴露于管腔内容物，并引发免疫反应和肠道炎症[20]，经PD治疗后，上述肠道炎症反应相关基因不同程度回调，表明PD有利于阻止DHD大鼠回肠上皮通透性的增加，维持回肠黏膜屏障功能.

    色氨酸代谢在炎症性肠炎中特别容易受到干扰[21]，KYNU(犬尿素酶)、TPH1(色氨酸羟化酶1)、KMO(犬尿氨酸3-单加氧酶)、IDO-1(吲哚胺2，3-双加氧酶)是色氨酸代谢相关通路的关键因子.Model组大鼠回肠组织TPH-1，KYNU，KMO，IDO-1的mRNA水平显著升高，表明DHD大鼠色氨酸代谢的2条途径即5羟色胺途径与犬尿氨酸都显著加强，色氨酸代谢通路被过度激活，经不同剂量的PD治疗后TPH-1，KYNU，KMO，IDO-1的mRNA水平极显著或显著降低，表明PD可调节回肠组织色氨酸代谢.

    MUC1，MUC2，ZO-1为紧密连接蛋白，是回肠黏膜屏障的重要组成部分[22]，黏蛋白构成黏液层内层，由上皮细胞分泌，是凝胶状分泌物的关键成分，具有调节组织黏膜屏障、参与细胞信号传导以介导机体免疫等生物学功能[23]，紧密连接蛋白ZO-1是肠黏膜免疫的关键蛋白，其基因表达量常被用于肠机械屏障功能的关键性检测标识[24]. 结合回肠组织形态学观察结果，Model组、SH组大鼠的回肠组织病理损伤严重，MUC1，MUC2的mRNA表达量急剧上升，ZO-1 mRNA的表达量显著下降，PD可显著降低肠黏膜损伤，PD组大鼠的MUC1，MUC2的mRNA表达量显著降低或降低，ZO-1 mRNA表达量显著上升.

    • 3.2.   结论

  • 本研究以“高糖高脂+高热高湿+肠毒性大肠杆菌”为诱导条件建立DHD大鼠模型，并以不同剂量的PD进行灌胃治疗.结果表明，经PD灌胃治疗后，大鼠回肠形态结构有所恢复，隐窝病变缓解.PD可以减轻大鼠IL-1β，IL-6，IL-17，TNFα，IFN-γ等促炎因子的生成，促进抑炎因子IL-4的生成来调节回肠炎症反应.PD可以降低TPH-1，KYNU，KMO，IDO-1的表达量从而阻止色氨酸代谢通路过度激活以调节色氨酸代谢，有效缓解DHD引起的肠道代谢紊乱.同时，PD阻止MUC1，MUC2升高、ZO-1降低从而使大鼠回肠黏膜屏障趋于稳态.

Figure (6)  Table (5) Reference (24)

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