The Microbiota-Dependent Treatment of Wuzhuyu Decoction for Chronic Migraine Model Rat Associated with Anxiety-Depression Like Behavior.

We previously found that Wuzhuyu Decoction (WZYD) could affect central and peripheral 5-HT to relieve hyperalgesia in chronic migraine (CM) model rats, possibly related to gut microbiota. However, the exact role of gut microbiota has not been elucidated. Accumulating evidence points to the possibility of treating central nervous system disease via the gut-brain axis. In our study, the inflammatory soup-induced CM model rats presented depression- and anxiety-like behaviors which both related to insufficient 5-HT. It was found that antibiotic administration caused community dysbiosis, and proteobacteria became the main dominant bacteria. The bacteria related to short-chain fatty acids and 5-HT generation were reduced, resulting in reduced levels of 5-HT, tryptophan hydroxylase, and secondary bile acids. Functional prediction-revealed sphingolipid signaling pathway in CM rats was significantly decreased and elevated after WZYD treatment. The effect of WZYD could be weakened by antibiotics. The CM rats exhibited anxiety- and depression-like behavior with 5-HT and number of neurons decreased in the CA1 and CA2 regions of hippocampal. The treatment of WZYD could recover to varying degrees. Antibiotics combined with WZYD attenuate the effect of WZYD on increasing the 5-HT content and related protein expression in the brain stem, plasma and colon, reducing CGRP, c-Fos, and inflammatory factors. And antibiotics also led to colon length increasing and stool retention, so that the antimigraine effect was weakened compared with WZYD. This experiment revealed that gut microbiota mediated WZYD treatment of CM rats with anxiety-depression like behavior.

Wuzhuyu Decoction relieves hyperalgesia by regulating central and peripheral 5-HT in chronic migraine model rats.

BACKGROUND: Chronic migraine (CM) is a highly disabling and burdensome disease. Wuzhuyu decoction (WZYD), a clinical used formula to treat and prevent episodic migraine and CM, has been reported to relieve the hyperalgesia of CM and increase brainstem and blood serotonin (5-hydroxytryptamine, 5-HT) in migraine model rats in previous studies; yet the mechanism is unclear. PURPOSE: This study aimed to observe the hyperalgesia relief effect of WZYD and investigate the mechanistic association with the regulation on central and peripheral 5-HT. METHODS: WZYD with different doses (3.372, 1.686 and 0.843 g/kg∙d) and the positive drug - sumatriptan (5.83 mg/kg∙3 d) were intragastrically administered in inflammatory soup (IS)-induced CM model rats, respectively. Hyperalgesia was assessed by facial mechanical withdrawal threshold and tail-flick latency. 5-HT was determined by ELISA. Western blot analysis, immunohistochemistry and immunofluorescence determination, and 16S rRNA gene sequencing were performed. RESULTS: WZYD significantly relieved the hyperalgesia by elevating the facial mechanical withdrawal threshold and tail-flick latency. In WZYD groups, increased 5-HT and decreased calcitonin gene-related peptide in both the brainstem and plasma, downregulated TNF-α, IL-1ß, and c-fos expression in the brainstem were observed in dose-dependent manner. Interestingly, 5-HT in colon tissues were also observed, which is associated with upregulating tryptophan hydroxylase, serotonin transporter and Piezo1 expression and increasing 5-HT and chromogranin A in enterochromaffin cells. Disorder of the microbiota, function and metabolism was correlated with 5-HT synthesis. WZYD could regulate the abundance of Anaerostipes and Acidifaciens. CONCLUSION: WZYD has the pharmacological effect on relieving hyperalgesia in CM model rats, possibly by affecting central and peripheral 5-HT.

[UPLC-MS/MS determination of active components from Wuzhuyu Decoction in plasma and brain tissues of nitroglycerin-induced migraine rats].

A sensitive and specific ultra-performance liquid chromatography-mass spectrometry(UPLC-MS/MS) method was deve-loped for analysis of rutaecarpine(Ru), evodiamine(Ev), rutaevine(Rv), limonin(Li), ginsendside Rb_1(Rb_1), ginsendside Re(Re) in rat plasma and brain tissues of nitroglycerin-induced migraine rats. Male healthy Sprague-Dawley(SD) rats were orally given multiple dose of optimized(OS) and un-optimized Wuzhuyu Decoction(UNOS), and their blood samples and brainstem were collected at different time points after injection of nitroglycerin(10 mg·kg~(-1)) into the frontal region. The drug concentrations of the 6 analytes in plasma and brainstem were determined by UPLC-MS/MS method. Subsequently, the main pharmacokinetics parameters of plasma were calculated by using Phoenix WinNolin 5.2.1 software. The methodological test showed that all of analytes in both plasma and brainstem homogenate exhibited a good linearity within the concentration range(r>0.994 7). The intra-day and inter-day accuracy, precision, matrix effect, stability of the investigated components meet the requirements for biopharmaceutical analysis. The developed method was successfully applied in pharmacokinetic studies on abovementioned ingredients in rat plasma and brain stem. The plasma pharmacokinetic parameters of active ingredients in two different Wuzhuyu Decoction group were compared, it was found that Rb_1 had higher t_(1/2), T_(max), C_(max), AUC_(0-24 h) and AUC_(0-∞ )in OS group. Meanwhile, Ev had higher t_(1/2) and T_(max) but lower C_(max), AUC_(0-24 h) and AUC_(0-∞), Ru has higher t_(1/2 )but lower C_(max), AUC_(0-24 h) and AUC_(0-∞ )in OS group. The brain tissue distribution of each component were compared between the two groups, the component with higher content in OS, such as Ru at 30 min and 2 h after administration, Ev at 30 min, Rb_1 at 30 min and Rb_1 at 2 h after administration have lower brain tissue distribution than those in UNOS group, while the component with higher content in UNOS, such as Rv at 30 min, 2 h and 12 h after administration had higher brain tissue distribution than those in OS group.

[Difference in intestinal absorption of Wuzhuyu decoction between normal and migraine model rats].

To compare the intestinal absorption of Wuzhuyu decoction(WZYD) between normal rats and migraine model rats, and investigate the optimized WZYD from aspect of absorption. The rat single pass intestinal perfusion test(SPIP) was adopted for optimized sample and un-optimized sample in normal and migraine model rats induced by nitroglycerin and reserpine. The contents of 8 ingredients were determined by high performance liquid chromatography(HPLC), and 4 absorption parameters for each ingredient were calculated and compared: unit area absorption(Mper area), absorption rate constant(Ka), apparent coefficient(Papp) and relative absorption rate(RA). The results showed that there was a great difference between normal rats and model rats in the intestinal absorption of the same WZYD. As compared with normal rats, the absorption parameters of most ingredients in optimized sample were increased in migraine model rats induced by nitroglycerin; Similar phenomena were also found in migraine model rats induced by reserpine. However, the absorption parameters of most ingredients were decreased in un-optimized sample. Therefore, pathological model rats shall be used for effective ingredient recognition based on the correlation between intestinal absorption spectra and pharmacological effects. As compared with the un-optimized samples, the absorption of effective ingredients was faster, easier and more adequate in the optimized samples, revealing their mechanism on better efficacy from the aspect of absorption.

[Study on correlation between color and effective components contents of Glycyrrhiza uralensis].

To explore the correlation between color of Glycyrrhiza uralensis and its quality evaluation,the colors of root bark and transverse section were determined by Precision Color Reader and Visual Analyzer,and the contents of six flavonoids and two saponins in G.uralensis were determined by high performance liquid chromatography（HPLC）.The partial least squares regression（PLSR）method was employed to correlate the colors with component contents in G.uralensis. The results showed that there were no significant differences in the colors of root bark but significant or very significant differences（P<0.05,P<0.01）in the colors of transverse section between the wild and cultivated G. uralensis. Compared with those in the cultivated G. uralensis, the contents of liquiritin, isoliquiritin isoliquiritigenin and the contents of ammonium glycyrrhizinate, glycyrrhetinic acid were obviously significant or remarkably significant in the wild G. uralensis.The correlation results showed that there was a significant or very significant correlation between the colors and the effective component contents. This study provides a scientific basis to evaluate the quality of G.uralensis by color and a new reference for the traditional evaluation methods for Chinese drugs.

[Study on intestinal absorption of ingredients from different compatibilities of Shaoyao Gancao decoction].

To study the compatible mechanisms and compatible proportion of Shaoyao Gancao decoction, the intestinal absorption of main ingredients in Shaoyao Gancao decoction SG11 (Baishao-Zhigancao 1: 1) , SG31 (Baishao-Zhigancao 3: 1), Baishao water decoction S and Zhigancao (G) were investigated and compared using in vitro everted intestinal sac model and in situ single pass intestinal perfusion (SPIP) model. The concentration of paeoniflorin (PF), liquiritin (LQ) and mono-ammonium glycyrrhizinate (GL) in test samples and samples of intestinal sac and intestinal perfusion was determined by HPLC. The intestinal absorptive amount and absorption parameters were calculated. Results showed that in the everted intestinal sac model, three ingredients could be absorbed by duodenum, jejunum and ileum, and the absorption in the jejunum was best for all 3 ingredients. The absorption rate of three ingredients in SG11 was significantly higher than that in single decoction (P < 0.05), but had no significant difference compared with SG31. In SPIP model, the absorption rate constant K(a), the apparent absorption coefficient P(app) and the absorption rate of three ingredients in SG11 were significantly higher than those in single decoction. Parameters of PF and GL in SG11 were significantly higher than those in SG31, but had no differences of LQ. It proved that the compatibility of Baishao and Zhigancao could improve the intestinal absorption of PF, LQ and GL. The absorption of each ingredient in SG11 was better than that in SG31.

[Study on pharmacological ingredients of wuzhuyu tang treating migraine by correlating absorption ingredients in everted intestinal sac and pharmacodynamics].

Wuzhuyu Tang is a classical formula for treating migraine, but its' pharmacological ingredients is unclear yet. Present study employed the everted intestinal sac model to collect the absorption samples of 10 kinds of Wuzhuyu decoction, and then analyzed the contents of 9 ingredients in Wuzhuyu Tang and absorption samples quantitatively or semi-quantitatively by HPLC-DAD method. Reserpine was used to establish the mice model of migraine, and then the contents and activities of 5-hydroxytryptamine, noradrenaline, dopamine, nitric oxide and nitricoxide synthase in brain tissues and serums were determined respectively after oral administration of Wuzhuyu Tang. Using the partial least squares regression method to correlate the total absorption quantity of 9 ingredients and pharmacodynamics. The result shows that limocitrin-3-O-beta-D-glucoside, ginsenoside Rg1 and Rb1, rutaevine, limonin, evodiamine and rutaecarpine are the main ingredients influenced the effects in absorption samples in everted intestinal sacs, especially ginsenoside Rg1, rutaevine, evodiamine and rutaecarpine among them have obvious improving effects to most pharmacodynamics index, might be the pharmacological ingredients influenced the therapeutical effects of Wuzhuyu Tang treating migraine.