Effects of Shu Gastric Formula modulating SCF/c-Kit pathway on gastric emptying function and interstitial cells of Cajal in rats with functional dyspepsia
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摘要: 目的 探讨舒胃方调控干细胞生长因子(stem cell factor，SCF)/c-Kit通路对功能性消化不良(functional dyspepsia，FD)大鼠胃排空功能及Cajal间质细胞(interstitial cells of Cajal，ICCs)的影响并进行分子对接，分析其作用机制。方法 32只SD级大鼠，随机取8只为空白组，另外24只通过多因素应激干预法建立FD大鼠模型，随机分为模型组、莫沙比利组及舒胃方组。光学显微镜观察大鼠胃窦组织；酚红法检测大鼠胃排空率；透射电镜观察大鼠ICCs的超微结构；ELISA法检测大鼠腹主动脉SCF含量；Western Blot检测大鼠胃窦组织SCF、c-Kit蛋白的表达；对舒胃方中所含活性成分进行收集与筛选，并对“有效成分-靶点”拓扑分析所得度值较高的有效成分进行分子对接。结果 各组大鼠胃壁组织结构完全，胃黏膜上皮完整，胃腺体结构和排列规整，未见明显炎性细胞浸润。空白组大鼠ICCs超微结构清晰，数量多，体积大，呈圆形、梭形，细胞核占比多，细胞质占比少，线粒体、内质网、核糖体多；模型组大鼠ICCs超微结构改变，数量少，体积小，线粒体肿胀或空泡化；莫沙比利组及舒胃方组大鼠ICCs超微结构改善，数量增多，体积变大，形态改善。与空白组比较，模型组大鼠胃排空率、血清SCF含量及胃窦组织SCF、c-Kit蛋白表达下降(P＜0.05)；与模型组比较，莫沙必利组与舒胃方组大鼠胃排空率、血清SCF含量及胃窦组织SCF、c-Kit蛋白表达增加(P＜0.05)。舒胃方中党参的主要活性成分有21个，炒白术的主要活性成分有7个，枳实的主要活性成分有22个，厚朴的主要活性成分有2个。分子对接结果显示，与关键靶点对接较好的成分有党参中的Daturilin，炒白术中的14-acetyl-12-senecioyl-2E，8Z，10E-atractylentriol，枳实中的nobiletin和厚朴中的Eucalyptol，与SCF、c-Kit的结合能分别为-6.44、-6.87、-5.72、-5.66、-4.31、-5.47、-5.69、-6.04。结论 舒胃方可能通过多成分、多靶点调控SCF/c-Kit通路，促进FD大鼠胃动力及ICCs增殖，调节胃肠功能。
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关键词:
- 分子对接 /
- 舒胃方 /
- SCF/c-Kit通路 /
- 功能性消化不良 /
- Cajal间质细胞
Abstract: Objective To investigate the effects of Shu Gastric Formula modulating the stem cell factor(SCF)/c-Kit pathway on gastric emptying function and interstitial cells of Cajal(ICCs) in functional dyspepsia(FD) rats and molecular docking, and to analyze its mechanism of action.Methods Thirty-two SD-grade rats, 8 were randomly taken as the blank group, and the other 24 were randomly divided into the model group, the mosapride group, and the Shu Gastric Formula group through the multifactorial stress intervention method to establish the FD rat model. The rat gastric sinus tissue was observed by light microscope; the gastric emptying rate was detected by phenol red method; the ultrastructure of rat ICCs was observed by transmission electron microscope; the content of serum SCF in the abdominal aorta of the rats was detected by ELISA; the expression of SCF and c-Kit protein in the gastric sinus tissue of the rats was detected by Western Blot; the active ingredients contained in Shu Gastric Formula were also analyzed by Western Blot; and the results were summarized. The active ingredients contained in Shu Gastric Formula were collected and screened, and molecular docking was carried out on the active ingredients with higher degree values obtained from the "active ingredient-target" topology analysis.Results The histological structure of the stomach wall of rats in each group was complete, the epithelium of gastric mucosa was intact, the structure and arrangement of gastric glands were regular, and no obvious inflammatory cell infiltration was seen. The ultrastructure of ICCs in rats of blank group was clear, with high number, large volume, round and pike shape, more nuclei, less cytoplasm, and more mitochondria, endoplasmic reticulum and ribosomes; the ultrastructure of ICCs in rats of model group was altered, with low number, small volume, and mitochondria were swollen or vacuolated; the ultrastructure of ICCs in rats of the mosapride group and the Shu Gastric Formula group was improved, with increased number, larger volume, and improved morphology. increased, the volume became larger, and the morphology improved. Compared with the blank group, the gastric emptying rate, serum SCF content, SCF and c-Kit protein expression of rats in the model group decreased(P < 0.05); compared with the model group, the gastric emptying rate, serum SCF content, SCF and c-Kit protein expression of rats in the mosapride group and the Shu Gastric Formula group increased(P < 0.05). Formula contained 21 key active ingredients of Codonopsis pilosula, 7 key active ingredients of Atractylodes macrocephala, 22 key active ingredients of Citrus aurantium, and 2 key active ingredients of Magnolia officinalis. The molecular docking results showed that the components that docked well with the key targets were Daturilin in Codonopsis, 14-acetyl-12-senecioyl-2E, 8Z, 10E-atractylentriol in Atractylodes macrocephala, nobiletin in Citrus aurantium, and Eucalyptol in Magnolia officinalis, the binding energies were -6.44, -6.87, -5.72, -5.66, -4.31, -5.47, -5.69, and -6.04 to SCF and c-Kit, respectively.Conclusion Shu Gastric Formula may promote gastric motility and proliferation of ICCs and regulate gastrointestinal function in FD rats through multi-component and multi-target regulation of SCF/c-Kit pathway. -
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表 1 各组大鼠胃窦组织SCF、c-Kit蛋白的表达
X±S 组别 例数 SCF蛋白 c-Kit蛋白 空白组 8 1.05±0.22 0.70±0.12 模型组 8 0.30±0.04 0.19±0.04 莫沙必利组 8 0.90±0.13 0.69±0.08 舒胃方组 8 0.90±0.13 0.69±0.11 F 36.152 57.836 P ＜0.001 ＜0.001 
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表 2 舒胃方的活性成分筛选
化合物ID 化合物名称 OB/% DL 所属中药 degree MOL008397 Daturilin 50.37 0.77 党参 88.87 MOL005828 nobiletin 61.67 0.52 枳实 87.67 MOL013433 prangenin hydrate 72.63 0.29 枳实 87.13 MOL001798 neohesperidin_qt 71.17 0.27 枳实 84.67 MOL008407 (8S，9S，10R，13R，14S，17R)-17-[(E，2R，5S)-5-ethyl-6-methylhept-3-en-2-yl]-10，13-dimethyl-1，2，4，7，8，9，11，12，14，15，16，17-dodecahydrocyclopenta[a]phenanthren-3-one 45.40 0.76 党参 83.40 MOL005321 Frutinone A 65.90 0.34 党参 82.90 MOL003036 ZINC03978781 43.83 0.76 党参 81.83 MOL000449 Stigmasterol 43.83 0.76 党参 81.83 MOL013352 Obacunone 43.29 0.77 枳实 81.79 MOL013435 poncimarin 63.62 0.35 枳实 81.12 MOL001006 poriferasta-7，22E-dien-3beta-ol 42.98 0.76 党参 80.98 MOL004355 Spinasterol 42.98 0.76 党参 80.98 MOL002140 Perlolyrine 65.95 0.27 党参 79.45 MOL013436 isoponcimarin 63.28 0.31 枳实 78.78 MOL000022 14-acetyl-12-senecioyl-2E，8Z，10E-atractylentriol 63.37 0.30 炒白术 78.37 MOL000028 α-Amyrin 39.51 0.76 炒白术 77.51 MOL013428 isosakuranetin-7-rutinoside 41.24 0.72 枳实 77.24 MOL006554 Taraxerol 38.40 0.77 党参 76.90 MOL005970 Eucalyptol 60.62 0.32 厚朴 76.62 MOL013440 citrusin B 40.80 0.71 枳实 76.30 MOL000021 14-acetyl-12-senecioyl-2E，8E，10E-atractylentriol 60.31 0.31 炒白术 75.81 MOL000033 (3S，8S，9S，10R，13R，14S，17R)-10，13-dimethyl-17-[(2R，5S)-5-propan-2-yloctan-2-yl]-2，3，4，7，8，9，11，12，14，15，16，17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol 36.23 0.78 炒白术 75.23 MOL006774 stigmast-7-enol 37.42 0.75 党参 74.92 MOL013276 poncirin 36.55 0.74 枳实 73.55 MOL000020 12-senecioyl-2E，8E，10E-atractylentriol 62.40 0.22 炒白术 73.40 MOL013277 Isosinensetin 51.15 0.44 枳实 73.15 MOL001803 Sinensetin 50.56 0.45 枳实 73.06 MOL008411 11-Hydroxyrankinidine 40.00 0.66 党参 73.00 MOL008391 5alpha-Stigmastan-3，6-dione 33.12 0.79 党参 72.62 MOL005980 Neohesperidin 57.44 0.27 厚朴 70.94 MOL009053 4-[(2S，3R)-5-[(E)-3-hydroxyprop-1-enyl]-7-methoxy-3-methylol-2，
3-dihydrobenzofuran-2-yl]-2-methoxy-phenol50.76 0.39 枳实 70.26 MOL004328 naringenin 59.29 0.21 枳实 69.79 MOL004492 Chrysanthemaxanthin 38.72 0.58 党参 67.72 MOL000049 3β-acetoxyatractylone 54.07 0.22 炒白术 65.07 MOL002879 Diop 43.59 0.39 党参 63.09 MOL008400 glycitein 50.48 0.24 党参 62.48 MOL007879 Tetramethoxyluteolin 43.68 0.37 枳实 62.18 MOL005100 5，7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-one 47.74 0.27 枳实 61.24 MOL008406 Spinoside A 39.97 0.40 党参 59.97 MOL013430 Prangenin 43.60 0.29 枳实 58.10 MOL013279 5，7，4'-Trimethylapigenin 39.83 0.30 枳实 54.83 MOL002914 Eriodyctiol(flavanone) 41.35 0.24 枳实 53.35 MOL008393 7-(beta-Xylosyl)cephalomannine_qt 38.33 0.29 党参 52.83 MOL007059 3-beta-Hydroxymethyllenetanshiquinone 32.16 0.41 党参 52.66 MOL003896 7-Methoxy-2-methyl isoflavone 42.56 0.20 党参 52.56 MOL007514 methyl icosa-11，14-dienoate 39.67 0.23 党参 51.17 MOL005849 didymin 38.55 0.24 枳实 50.55 MOL000006 luteolin 36.16 0.25 党参 48.66 MOL000006 luteolin 36.16 0.25 枳实 48.66 MOL000072 8β-ethoxy atractylenolide Ⅲ 35.95 0.21 炒白术 46.45 MOL013437 6-Methoxy aurapten 31.24 0.30 枳实 46.24 MOL001941 Ammidin 34.55 0.22 枳实 45.55
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表 3 舒胃方中活性成分与靶点的结合能
活性成分 靶点 结合能 蛋白选择 党参中的Daturilin SCF -6.44 P21583 党参中的Daturilin c-Kit -6.87 P10721 炒白术中的14-acetyl-12-senecioyl-2E，8Z，10E-atractylentriol SCF -5.72 P21583 炒白术中的14-acetyl-12-senecioyl-2E，8Z，10E-atractylentriol c-Kit -5.66 P10721 枳实中的nobiletin SCF -4.31 P21583 枳实中的nobiletin c-Kit -5.47 P10721 厚朴中的Eucalyptol SCF -5.69 P21583 厚朴中的Eucalyptol c-Kit -6.04 P10721
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