Huangqi Guizhi Wuwu Decoction Ameliorates Focal Cerebral Ischemia-Reperfusion Injury in Rats: Pharmacological and Metabolomics Evidences
doi: 10.14148/j.issn.1672-0482.2021.0920
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摘要:
目的 研究黄芪桂枝五物汤对脑缺血损伤的神经保护作用，并探讨其可能作用机制。 方法 将50只大鼠随机分为假手术组, 模型组及黄芪桂枝五物汤低、中、高剂量组(5、10、20 g·kg-1)，每组10只。除假手术组外，大鼠采用线栓法建立大脑中动脉闭塞(MCAO)模型。中药组大鼠灌胃给予复方水煎液，假手术组和模型组大鼠灌胃给予等量的生理盐水。连续灌胃7 d后，处死大鼠。通过神经功能缺损评分、脑梗死面积、脂质过氧化和炎症细胞因子水平检测来评估各组大鼠脑缺血性损伤程度。同时通过代谢组学研究黄芪桂枝五物汤对脑缺血再灌注损伤的可能机制。 结果 黄芪桂枝五物汤可显著降低神经功能缺损评分和脑梗死面积，减少脂质过氧化和炎症细胞因子水平，表明其可有效减轻MCAO诱导的大鼠脑缺血性损伤。进一步的代谢组学研究表明，脑缺血再灌注损伤后血清代谢紊乱，共筛选和鉴定出23种与缺血性中风相关的代谢物发生显著性变化，是脑缺血潜在的生物标志物。代谢通路分析显示，MCAO主要通过靶向乙醛酸和二羧酸代谢，以及苯丙氨酸、酪氨酸和色氨酸的生物合成来诱导脑缺血性损伤。黄芪桂枝五物汤干预后，大部分代谢标记物发生了逆转, 主要包括吲哚氧基硫酸、柠檬酸、3-羟基十二酸、3-甲基-2-丁烯酸、苹果酸、丁醛和尿酸等。 结论 黄芪桂枝五物汤可有效改善大鼠局灶性脑缺血再灌注损伤, 其发挥神经保护作用的机制可能与抑制炎症、改善多种代谢途径有关。 Abstract:OBJECTIVE To investigate the neuroprotective effect of Huangqi Guizhi Wuwu Decoction (HGWD) on cerebral ischemia injury, and to explore the underlying mechanism. METHODS Fifty rats were randomly divided into 5 groups: sham operation group (sham), middle cerebral artery occlusion (MCAO) model group, and MCAO rats treated with HGWD at 5, 10, 20 g·kg-1. The first two groups were administered intragastrically (i.g.) with saline for 7 days, while HGWD groups were treated with HGWD at 5, 10, and 20 g·kg-1, correspondingly. The neurological deficits scores, cerebral infarct size, lipid peroxidation and inflammatory cytokines levels were used to evaluate the brain ischemic injury. The possible mechanism of HGWD on cerebral ischemia-perfusion injury was investigated by metabolomics. RESULTS HGWD treatment significantly attenuated middle cerebral artery occlusion (MCAO)-induced brain ischemic injury of rats as evidenced by decreasing the neurological deficits scores, cerebral infarct size, as well as lipid peroxidation and inflammatory cytokines levels. Further metabolomics study indicated that great metabolic disorders in serum were induced following cerebral ischemia/reperfusion injury. Totally 23 metabolites associated with ischemic stroke were proposed as potential biomarkers. The metabolic pathway analysis revealed that MCAO induced brain ischemic injuries mainly by targeting glyoxylate and dicarboxylate metabolism, and phenylalanine, tyrosine and tryptophan biosynthesis. Correspondingly, HGWD could restore most of the imbalanced endogenous metabolites. CONCLUSION HGWD plays a protective role in focal cerebral ischemia-reperfusion injury in rats, which may be associated with the inhibition of inflammatory effect and improvement of multiple metabolic pathways. -
Figure 2. The effect of HGWD on scores of neurological deficit and cerebral infarction size in rats
Note: (A) Neurological deficit scores were determined according to Zea-Longa 5-point scheme. (B) TTC staining photograph in Sham, MCAO, HGWD groups. (C) infarct volume.
*P < 0.05, **P < 0.01, compared with MCAO group. ##P < 0.01, compared with sham group.
Figure 3. Degeneration of brain tissues and neurons in MCAO group, ameliorated by HGWD pretreatment
Figure 4. Restoring effects of HGWD on biological parameters and expression of inflammatory factors in brain of rats
Note: ##P < 0.01, compared with the control group; *P < 0.05, * *P < 0.01, compared with the MCAO group.
Figure 5. PLS-DA Plots of metabolic disturbances of MCAO-induced focal cerebral ischemia
Note: The PLS-DA plots showed clear separation between Control and MCAO. (A) Positive: R2X=0.791, Q2=0.653. (B) Negative: R2X=0.557, Q2=0.575.
Figure 6. Metabolites variation analysis
Note: (A) Heatmap of metabolite concentrations. (B) Metabolism pathway affected by MCAO treatment.
a. Arginine and proline metabolism; b. Citrate cycle (TCA cycle); c. Glyoxylate and dicarboxylate metabolism; d. Phenylalanine, tyrosine and tryptophan biosynthesis; e. Glycerophospholipid metabolism.
Figure 8. Interaction networks of enzymes and proteins
Note: (A) Functional interaction networks of the key enzymes were collected and input into the STRING database to analyze interactions between them (interaction score=0.9). Acly, ido1, mdh2 and tph1 were 4 most interactive enzymes. Functional interaction networks of acly (B), ido1 (C), mdh2 (D) and tph1 (E). Only proteins with highest confidence (interaction score=0.9) were considered as possible targets network for the metabolites.
Table 1. Significantly differential metabolites
HMDB Code Name m/z Retention
time/minP-value VIP FC (+) HMDB0000509 3-Methyl-2-butenoic acid 101.059 72 0.69 0.001 948 2.051 59 1.33 HMDB0000714 Hippuric acid 180.065 42 7.06 0.001 948 1.109 93 4.00 HMDB0003543 Butanal 73.064 54 17.65 0.003 876 1.228 98 1.19 HMDB0000158 L-Tyrosine 182.081 14 3.59 0.003 876 3.816 20 1.85 HMDB0010395 LysoPC(20∶4 (5Z, 8Z, 11Z, 14Z)) 544.338 45 15.83 0.003 876 6.214 17 1.65 HMDB0002302 Indole-3-propionic acid 190.086 11 10.81 0.005 385 1.245 83 3.75 HMDB0000517 L-Arginine 175.118 84 1.44 0.007 406 1.582 33 1.58 HMDB0010383 LysoPC(16∶1(9Z)/0∶0) 494.323 09 15.35 0.010 082 3.642 14 2.59 HMDB0001080 4-Aminobutyraldehyde 88.075 50 15.20 0.013 587 3.478 89 2.04 HMDB0000734 Indoleacrylic acid 188.070 50 5.81 0.013 587 1.221 11 1.33 HMDB0000387 3-Hydroxydodecanoic acid 217.179 71 16.94 0.013 587 2.288 45 1.91 HMDB0000929 L-Tryptophan 205.097 11 5.81 0.0181 29 3.436 01 1.23 HMDB0000097 Choline 104.106 89 1.48 0.023 949 2.027 37 1.43 HMDB0010404 LysoPC(22∶6(4Z, 7Z, 10Z, 13Z, 16Z, 19Z)) 568.338 35 15.76 0.031 324 1.485 42 1.33 - 1, 4-Dioxane 89.059 53 17.21 0.000 939 2.152 93 1.37 (-) HMDB0001865 2-Ketovaleric acid 115.039 56 4.84 0.000 939 2.459 03 3.14 HMDB0001864 2-Ketohexanoic acid 129.055 70 7.25 0.000 939 5.719 27 3.04 HMDB0000094 Citric acid 191.018 08 2.62 0.000 939 2.473 79 2.48 HMDB0000943 Threonate 135.029 11 2.44 0.005 385 7.506 99 1.60 HMDB0000744 Malic acid 133.013 58 1.83 0.007 406 2.119 47 2.53 HMDB0000289 Uric acid 167.019 83 2.51 0.010 082 2.490 11 1.55 HMDB0000682 Indoxylsulfuric acid 211.999 98 6.89 0.010 082 1.768 79 2.11 HMDB0003345 α-D-Glucose 179.054 76 2.44 0.023 949 3.301 15 1.51 
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Table 2. Key enzymes of HGWD treatment
Enzyme Full name Uniprot ID Glyat Glycine N-acyltransferase Q6IB77 Glyatl3 Glycine N-acyltransferase-like protein 3 Q5SZD4 Ddc Aromatic-L-amino-acid decarboxylase P20711 Ido1 Indoleamine 2, 3-dioxygenase 1 P14902 Tph1 Tryptophan 5-hydroxylase 1 P17752 Acly ATP-citrate synthase P53396 Akr1b1 Aldose reductase P15121 Mif Macrophage migration inhibitory factor P14174 Ugt1a1 UDP-glucuronosyltransferase 1-1 P22309 Me1 NADP-dependent malic enzyme Q16798 Me2 NAD-dependent malic enzyme P23368 Rdh13 Retinol dehydrogenase 13 Q8NBN7 Hk2 Hexokinase-2 P52789 Enpp3 Ectonucleotide pyrophosphatase/Phosphodiesterase family member 1 P22413 Hk1 Hexokinase-1 P19367 Gck Glucokinase P35557 Hk3 Hexokinase-3 P52790 Mdh2 Malate dehydrogenase P40925
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Table 3. Key proteins of HGWD
Enzyme Full name Uniprot ID Acaca Acetyl-CoA carboxylase 1 Q13085 Dhtkd1 Dehydrogenase E1 and transketolase domain containing 1 Q96HY7 Mdh1 Malate dehydrogenase, cytoplasmic P40925 Mdh2 Malate dehydrogenase, mitochondrial P40926 Ogdh 2-Oxoglutarate dehydrogenase, mitochondrial Q02218 Ogdhl 2-Oxoglutarate dehydrogenase-like, mitochondrial Q9ULD0 Sdha Succinate dehydrogenase[ubiquinone]flavoprotein subunit, mitochondrial P31040 Sdhb Succinate dehydrogenase[ubiquinone]iron-sulfur subunit, mitochondrial P21912 Sucla2 Succinyl-CoA ligase Q9P2R7 Afmid Arylformamidase Q63HM1 Ddc Aromatic-L-amino-acid decarboxylase P20711 Kynu Kynureninase Q16719 Maoa Amine oxidase P21397 Tdo2 Tryptophan 2, 3-dioxygenase P48775 Tph1 Tryptophan 5-hydroxylase 1 P17752 Tph2 Tryptophan 5-hydroxylase 2 Q8IWU9 Acly ATP-citrate synthase isoform 1 P53396 Atp5c1 ATP synthase subunit gamma, mitochondrial P36542 Cs Citrate synthase O75390 Fh Fumarate hydratase, mitochondrial precursor P07954 Got1 Aspartate aminotransferase, cytoplasmic P17174 Got2 Aspartate aminotransferase, mitochondrial P00505 Me1 NADP-dependent malic enzyme P48163 Me2 NAD-dependent malic enzyme, mitochondrial P23368 Me3 NADP-dependent malic enzyme, mitochondrial Q16798 Ido1 Indoleamine 2, 3-dioxygenase 1 P14902 Ido2 Indoleamine 2, 3-dioxygenase 2 Q6ZQW0 Il4i1 Interleukin 4 induced 1 Q96RQ9
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