The bio-active components of the Mongolian medicine Horcha-6 and therapeutic mechanism in the rat migraine model

Background: The active components of Horcha-6 were identified using liquid chromatography with tandem mass spectrometry. Also, we investigated the potential mechanisms that explain why Horcha-6 may be effective in treating migraines through the use of network pharmacology and a rat migraine model. Methods: After identifying the active components of Horcha-6, the corresponding genes of the active components’ target were obtained from the Universal Protein database, and a “compound-target-disease” network was constructed using Cytoscape 3.9.0 software. For the in vivo experiments, nitroglycerin was injected intraperitoneally into rats to create a migraine model. Pre-treatment with Horcha-6 was administered orally for 14 days, and rats were subjected to migraine-related behavior tests. RNA sequencing was performed to identify the gene expression regulated by Horcha-6 in the trigeminal nerve. Results: A total of 903 chemical components of Horcha-6 have been collected in the liquid chromatography with tandem mass spectrometry. We discovered 55 of the Horcha-6 bio-active components that were evaluated based on their Percent Human Oral Absorption (≥ 30%) and DL values (≥ 0.185) on the traditional Chinese medicine systems pharmacology database. The “compound-target-disease” network contained 163 intersection targets with the migraine state. Gene Ontology analysis indicated that these components significantly regulated the immune response, vascular function, oxidative stress, etc. When Kyoto Encyclopedia of Genes and Genomes enrichment analysis was performed, we observed that most of the target genes were significantly enriched in the inflammation and neuro-related signaling pathway, toll-like receptor signaling pathway, neuroactive ligand-receptor interaction, etc. These predictions were further demonstrated via in vivo animal model experiments. The RNA sequencing results showed that 41 genes were down-regulated ( P < 0.05) and 86 genes were up-regulated ( P < 0.05) in the Horcha-6 treated group compared with the untreated group. Those genes were mainly involved in neuromodulation, vascular function, and hormone metabolism. Conclusion: The 55 bio-active components in Horcha-6 regulate


Background
Migraine is the prevailing neurological disorder in terms of prevalence.The syndrome is characterized by episodes of moderate to severe headaches and intermittent neurological and systemic symptoms [1].Epidemiological investigations have shown that a migraine affects over 1 billion people worldwide and is almost three times more common in women than in men [2].This prevalence and associated disability have caused many burdens on society and families.The occurrence of migraines affects not only the individuals' physical health but also the interpersonal dynamics, psychological health, and financial stability of the entire family.Therefore, an increasing number of researchers are investigating the mechanism and treatment of migraines [1,2].
Although the physiological mechanisms of the development of migraines are still not completely understood, a common conception suggests that the chronic migraine is a threshold disorder, which means that the sensory threshold decreases and susceptibility to attack-inducing stimuli increases [3].Migraines can be initiated by various factors such as genes, environment, metabolism, hormones, and drugs [4].With these repetitive nociceptive stimulations, the trigeminal nerve and descending pain-modulating system, including the periaqueductal gray, are activated.Frequent migraine attacks ultimately lead to oxidative stress and dysfunction of the descending pain-modulating network [5].These dysfunctions, in turn, might increase the susceptibility to attack-inducing stimuli and subsequently lower the sensory threshold.If not treated on time, it would be developed into chronic migraine.
Inflammation plays an important role in the initiation and progression of various neurological disorders [6].A systemic review and mate-analysis indicate that levels of the pro-inflammatory factors IL-6, IL-8 and TNF-α were higher in migraine patients than in the normal people, and IL-1β levels were also higher during the attack [7].These cytokines are related with infection, fluid shear stress, atherosclerosis, and TNF signaling pathways.Mechanical stimulation of fluid flow has also thought to be a key factor in regulating the immune activity of microglia in brain diseases [8].Furthermore, a genome-wide association study summary statistics found that higher levels of Dickkopf1 and platelet derived growth factor subunit B, as well as lower levels of phenylalanyl-TRNA synthetase 2, glutathione s-transferase alpha 4 and cysteine rich hydrophobic domain 2 proteins, had a significant causal relationship with migraine [9].It suggests that β-catenin-dependent Wnt signaling is down-regulated in migraine.Therefore, the mechanism of chronic migraine is complicated, and identifying an appropriate, beneficial treatment is challenging.
Current management of migraines includes recognizing and eliminating the specific exacerbating factors, and preventive and acute treatment.In clinics, triptans, non-steroidal anti-inflammatory drugs, and antiemetics are used for acute migraine therapy, while beta-blockers, tricyclic antidepressants, and anticonvulsants are used as preventive therapy.However, there are no radical treatment methods for migraines.Horcha-6 (Norcheg-6, Marcheg-6, Yama-6, Liu-Rui Wan) is a classic preparation of traditional Mongolian medicine (TMM).It was first recorded in "Obed's on Dalai" that is a masterwork of TMM written in 1977 [10].Horicha-6 has been used clinically ever since and mainly to treat a variety of migraines.Clinical observation revealed that Horcha-6 had the greatest effect on treating migraines caused by upper respiratory tract infections like rhinitis, pharyngitis, and tonsillitis [11].
These previous studies and literature indicate that Horcha-6 may have anti-neuroinflammatory properties.However, as a polyherbal formulation, the mechanism of action of Horcha-6 remains unclear.In this study, we aimed to clarify the potential mechanisms of Horcha-6 on migraine therapy by network pharmacological approach associated with RNA sequencing.

Animal models and treatment
Wistar rats (female, 8 weeks old, 200 ± 20 g) were purchased from SiPeiFu Co., Ltd.(Beijing, China).Rats were maintained in a normal environment on a 12-h light/dark cycle (lights on 06:00-18:00 h) with ad libitum access to food and water and were fed a standard diet.This study was conducted by the National Institutes of Health Guide for the Care and Use of Laboratory Animals and was approved by the Institutional Animal Care and Use Committee of Inner Mongolia University (ethics approval number No. IMU-2022-Rat-049).
After acclimatization and adaptive feeding for 5 days, a total of 60 rats were allocated into five groups, namely Control, Model, Sumatriptan, Horcha-6 low (HRC-6-L), and Horcha-6 high (HRC-6-H), using a random assignment method.The total duration of this experiment was 15 days, during which the control group received no treatment; the model group was given saline (2 mL) daily; the Sumatriptan group was given Sumatriptan (25 mg/kg) daily; the HRC-6-L group was given Horcha-6 (310 mg/kg) daily; and the HRC-6-H group was given Horcha-6 (620 mg/kg) daily.In addition, rats of each group except for the control group, received an intraperitoneal injection of nitroglycerin (10 mg/kg) on the 7th and 15th day of the experiment.
Preparing drugs and dose calculation: Horcha-6 was purchased from a drugstore (Z15020284, Inner Mongolia Huriyee Mongolian Medicine Co., Ltd., Tongliao, China).The recommended dosage for the clinical administration of Horcha-6 in adults with a body weight of 60 kg is 3 g each dose, which corresponds to a dosage of 50 mg/kg of body weight.According to body surface area, the ratio of equivalent dose

Highlights
Horcha-6 can relieve migraines through its bio-active components, such as quercetin, luteolin, and kaempferol, to modulate fluid shear stress, vascular smooth muscle contraction, TGF-β, Hippo, and Wnt signaling pathways, which directly related the initiation and development of migraine.

Medical history of objective
Horcha-6, a classical formula used for depression, is derived from Zhanbula-Quejisenfurenlai's "Obed's on Dalai" (18th-19th century).In ancient times, Horcha-6 was often used for It has the functions of reducing "Nian" (inflamation), clearing "Xila" (one element of three elements of life in Mongolian medicine theory, Symptoms: Headache, stomach, thirst, fever, local pain, thick tongue coating, yellow urine with a strong smell, and arrhythmia), and relieving pain; Mainly used for "Xila" type headaches, red and swollen eyes, and side headaches caused by "Yama" (rhinitis).Submit a manuscript: https://www.tmrjournals.com/tmrbetween humans and rats is determined to be 6.2.Therefore, the Horcha-6 dose given to rats per kg of body weight was 310 mg in the HRC-6-L group (Formula: Horcha-6 50 mg/kg × 6.2 = 310 mg/kg) and 620 mg in the HRC-6-H group (Formula: Horcha-6 310 mg/kg × 2 = 620 mg/kg).In the Sumatriptan group, mice had previously received Sumatriptan orally at 600 μg/kg [16].Consequently, following the computation of the dosage for each rat, the medicines were subsequently dissolved in a 2 mL solution of filtered water.The administration of all treatments performed orally on a daily basis at a consistent time.

Detection of rat behavior
During the experiment, we observed the rat behavior changes in each group after intraperitoneal injection of nitroglycerin, which is similar to a previous study [17].After intraperitoneal injection of nitroglycerin, we observed rats' spontaneous nociceptive behavior, light-aversive behavior, and spontaneous tactile allodynia.The spontaneous nociceptive behavior that was record by Camera in clear acrylic device including exploration, locomotion, freezing, grooming, and resting behavior.The light-aversive behavior was performed as in the literature (for details, see Esposito, E. et al.) [18].Then we obtained spontaneous tactile allodynia after intraperitoneal injection of nitroglycerin for 120 min, the rat was placed in the center of the clear acrylic device and allowed to move freely between rough and smooth surface, which recorded 5 mins by camera and analyzed the residence time (for details, see Bolay, H. et al.) [19].The rat behavior detection process is shown in Figure 1A.In addition, each of the tests required a different device, as follows: The clear acrylic installation: this device consists of a clear acrylic chamber (45 × 45 × 35 cm 3 ), in which rats were able to move freely.The light/dark chamber: this device consists of two identical chambers (30 × 30 × 22.5 cm 3 ), the light room is placed in a bright room without a lid, and the dark room is total black and covered with a lid.A small door (10 × 10 cm) connects the two chambers, and rats were able to move freely.
The rough/smooth surface device: this device consisted of a transparent acrylic box (45 × 45 × 35 cm 3 ), in which the floor is divided into two equal fields (each area: 22.5 × 45 cm) where the left floor surface is covered with smooth sandpaper (P1000 grit) and the right floor surface is covered with rough sandpaper (P40 grit).The rats had free access to move between the fields.

Liquid chromatography with tandem mass spectrometry (LC-MS/MS)
Metabolite extraction: 100 mg of Horcha-6 sample was added to 500 mL of 80% methanol solution containing 0.1% formic acid.The samples were heat shocked and placed in ice-water for five min.After resting, the samples were centrifuged at 15,000 rpm for 10 min at 4 °C.A certain amount of the supernatant was added to mass spectrometry-grade water to dilute the methanol solution to 53% and was centrifuged at 15,000 rpm for 10 min at 4 °C.This supernatant was used for LC-MS/MS analysis.The QC sample was a mixture of an equal volume of each experimental sample.The blank sample was the 80% methanol solution containing 0.1% formic acid, and all were processed in the same manner as the experimental samples.
A Thermo QE mass spectrometer in conjunction with the CD database search program to get the MS and tandem MS/MS database.Within each acquisition cycle, the mass range measured fell within the range of 70 to 1,050.The MS/MS secondary scans were data-dependent scans.The specifications were as follows: sheath gas flow rate, 35 Arb; aux gas flow rate, 10 Arb; capillary temperature, 320 °C; and spray voltage, 3.2 kV.

Network analysis
The chemical components of Horcha-6 were determined using LC-MS/MS.The screening process involved evaluating the percent human oral absorption rate, which needed to be equal to or greater than 30%, as well as assessing the DL value, which needed to be equal to or greater than 0.185.The chemical targets of Horcha-6 were acquired from the traditional Chinese medicine systems pharmacology database and analysis platform database (https://old.tcmsp-e.com/tcmsp.php)[20].The disease targets of migraines were obtained from the Gene Cards Database (https://www.genecards.org),National Center of Biotechnology Information database (https://www.ncbi.nlm.nih.gov/), and Online Mendelian Inheritance in Man database (https://omim.org/)[21][22][23].The UniProtKB search function of the UniProt database (http://www.uniprot.org/)was used to standardize the names of the predicted target genes [24].Finally, Venn analysis was performed on the Horcha-6 and migraine target genes to obtain the common target genes.These target genes were used for the Gene Ontology (http://www.geneontology.org/)[25] and Kyoto Encyclopedia of Genes and Genomes pathway (www.kegg.jp/kegg/pathway.html)analysis, P < 0.05 indicates significant differences [26].Finally, using Cytoscape 3.9.0 a protein-protein interaction network was constructed for the main pathway related genes and metabolic components.

RNA sequencing
After the last observation, the rats were killed under anesthesia, dissected the trigeminal nerve, and immediately frozen at −80°C.Total RNA was extracted using trizol (Ambion/invitrogen, Carlsbad, California, USA), and RNA integrity was accurately detected by Ailent2100 bioanalyzer.The mRNA was then obtained, the cDNA was synthesized, and the gene fragment was sequenced using Illumina (NovaSeq 6000, Hayward, USA) platform.The raw reads of fastq format were filtered by removing adapter and low-quality reads that contained poly-N sequences or inferior quality reads to obtain clean reads.HISAT2 software was used to quickly and accurately compare the Clean Reads with the reference genome to obtain the location information of Reads on the reference genome.The StringTie software is then used to assemble the new transcript.The expression levels of genes in each sample were estimated using the DESeq2 R Package (1.16.1), and the P-value and fold change were calculated.Genes with |Log2FC| ≥ 1 and an adjusted P < 0.05 were assigned as differentially expressed genes.The volcano plots and heatmap were generated with MetwareCloud online platform.The Metascape database was used to perform the GO and KEGG enrichment analysis of the differentially expressed genes, and the pathways with a P-value below 0.05 were considered significantly enriched.

Statistical analysis
All data are represented as mean ± standard error of the mean.The statistical significance was assessed by using a one-way analysis of variance, followed by Dunnett's multiple comparisons, where considered appropriate (GraphPad Prism 7, La Jolla, California, USA).Statistical significance has been assigned to results with a P-value less than 0.05.

Horcha-6 components
Traditional prescriptions featured an unusual feature of integrating many components and targeting multiple pathways, becoming an interesting difficulty for pharmacological investigations.Thus, the detailed TMM chemical components of Horcha-6 are important for their pharmacological study.In this study, we determined the components of Horcha-6 through the use of LC-MS/MS.As illustrated in Figure 2A, a total of 354 chemical components have been obtained using the negative electrospray ionization mode of LC-MS/MS.There were 199 components classified as lipids and lipid-like molecules, organic acids and derivatives, benzenoids, phenylpropanoids and polyketides, organoheterocyclic compounds, and organic oxygen compounds (Figure 2B).The KEGG enrichment analysis indicated that these components were mainly related to the metabolism of the global and overview maps, amino acid metabolism, biosynthesis of other secondary metabolites, and carbohydrate metabolism (Figure 2C).In the positive electrospray ionization mode of LC-MS/MS, we obtained 549 components, of which 249 components most were lipids and lipid-like molecules, organoheterocyclic compounds, organic acids and derivatives, benzenoids, and phenylpropanoids and polyketides (Figure 2D, 2E).The KEGG enrichment analysis indicated that these components were mainly related to the metabolism of the global and overview maps, amino acid metabolism, biosynthesis of other secondary metabolites, and nucleotide metabolism (Figure 2F).These compounds displayed a broad range of bioactivities, including anti-inflammatory, immunostimulatory, anti-cancer, antioxidant, and antimicrobial activities [27].These results indicated that Horcha-6 has multiple potential pharmacological activity components.

Relationship between Horcha-6 components and migraines
The network analysis "Compound-Target-Disease" is appropriate to explore the complicated TMM therapeutic effects of multiple Submit a manuscript: https://www.tmrjournals.com/tmrcompounds and targets [28].Therefore, we performed a network analysis of the Horcha-6 metabolites that had been detected by the LC-MS/MS assay to explore the pharmacological actions of Horcha-6.We obtained 55 components of Horcha-6 from the traditional Chinese medicine systems pharmacology database and analysis platform database, which were subjected to screening based on the specifications of having a percent human oral absorption of at least 30% and DL values of at least 0.185 (Table 1).Meanwhile, there were 277 targets genes related to the Horcha-6 components.There were 3,015 migraine target genes obtained from the Gene Cards, National Using the Venn analysis, we identified 163 common target genes among the Horcha-6 and migraine-related target genes (Figure 3A).The top 10 target genes identified from the screening values were PTGS2, PTGS1, HSP90AA2P, NCOA2, SCN5A, ADRB2, CHRM1, CAMK1G, DPP6, and AR.Enrichment analyses using GO and KEGG have been conducted on a total of 163 target genes.The 277 Horcha-6 gene targets for migraine treatment were introduced into String 11.5 to construct a PPI network and the top 20 key genes were screened out according to their degree value (AKT1, TP53, TNF, JUN, IL6, VEGFA, CASP3, MAPK3, ESR1, HIF1A, MYC, PTGS2, IL1B, PPARG, PTEN, CCND1, MMP9, EGF, FOS and ERBB2) [29].GO analysis indicated that these components significantly regulated the immune response, vascular function, oxidative stress, cell signaling transduction, and cell structure like membrane and synapse (Figure 3B).In the KEGG enrichment analysis, most of the target genes were significantly enriched in the inflammation, atherosclerosis, and neuro-related signaling pathways, such as lipid and atherosclerosis, TNF signaling pathway, toll-like receptor signaling pathway, and neuroactive ligand-receptor interaction.(Figure 3C).To further understand the relationship between Horcha-6 and these signaling pathways, a PPI network was constructed of the top 20 KEGG pathway enriched genes and related Horcha-6 components.As shown in Figure 3D, there were 117 target genes and 50 metabolites related to these signaling pathways.These findings suggest that Horcha-6, through regulating the inflammatory reaction, and vascular and neuro system function, can relieve migraines.

Horcha-6 relieved the abnormal behavior of rat migraine models
The results of the clinical efficacy, metabolites, and network analysis indicate that Horcha-6 has a therapeutic effect on migraines.Therefore, in this experiment, we constructed a rat migraine model using nitroglycerin to reveal the potential therapeutic effects of Horcha-6.Numerous studies have demonstrated that nitroglycerin triggers cranial allodynia associated with migraine-like headaches and appears to have a similar phenotype [30].During the construction of the model, we observed that the body weight changes of rats in each group were not significantly different (ns, P > 0.05, Figure 1B).On the 7th and 15th day of the experiment, we injected nitroglycerin into the rats of each group except for the control group and observed the abnormal behaviors.In the first test, we observed that the model rat behaviors like exploration, freezing, locomotion, resting, and light aversion differed from that of the control group.In the Horcha-6 treatment groups, the behaviors of freezing and grooming were significantly decreased, and the exploratory behavior was increased compared with the model group ( * P < 0.05, ** P < 0.01, Figure 1C).In Figure 3 Network analysis to discover the association between the Horcha-6 components and migraines.The Venn diagram illustrates the relationship between the target genes of the Horcha-6 compounds and genes associated with migraines.A total of 163 target genes were discovered with regard to each group.The GO enrichment analysis was performed on the 163 target genes, with a focus on the top 10 pathway enrichments in every group of biological process, cellular components, and molecular function.The KEGG was used to perform enrichment analysis on a set of 163 target genes.The top 20 pathways exhibiting enrichment were identified.The present study examines the relationship between each of a set of 118 genes that exhibited enrichment in the top 20 signaling pathways as defined by the KEGG, and the components of Horcha-6.The nodes in the graphical representation are color-coded, with green denoting Horcha-6 compounds and blue representing associated genes.KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology.Submit a manuscript: https://www.tmrjournals.com/tmrthe second test, similar results were observed with the greatest behavioral differences being observed between the control and model group.These behaviors were significantly relieved in the treatment groups (sumatriptan and Horcha-6) compared with the model group.Moreover, there was no differences in rat behavioral changes between the treatment groups of Sumatriptan (positive treatment control) and Horcha-6 (Figure 1D).These results indicated that Horcha-6 relieved the abnormal behavior of the rats resulting from the migraine model induced by nitroglycerin.
Horcha-6 reduced oxidative stress, regulated the vascular function, and improved cellular regeneration and repair in the migraine model rats' trigeminal neuralgia Presently, it is believed that the initiation of a migraine is closely related to the trigeminal [31].These results illustrate that the trigeminal is a key target for migraine treatment.Other studies indicated that nitroglycerin administration not only produces attacks which are phenotypically similar to spontaneous migraine attacks and sensitizes the trigeminal and cortical structures [32].In order to comprehensively explain the regulatory effects of Horcha-6 on the trigeminal neuralgia of rats in a migraine model, we carried the transcriptome investigation of the trigeminal neuralgia.As shown in Figure 4A, we found the down-regulated genes of the treatment groups, and these genes were up-regulated in the model group.We compared these 41 down-regulated genes through Venn analysis and found that the Horcha-6-H had the most significant effect.Therefore, we performed GO analysis on these 41 down-regulated genes and found the most genes were related to metabolism and development, such as small molecule catabolic process, embryonic organ development, fatty acid beta-oxidation, and nerve development (Figure 4B).In the KEGG enrichment analysis, we found these genes were significantly enriched in metabolism, neuromodulation, and vascular function, such as fatty acid metabolism, neuroactive ligand-receptor interaction, fluid shear stress and atherosclerosis (Figure 4C).The generated heat map indicated the condition of these 41 genes expressed in each group (Figure 4D).Furthermore, we thoroughly analyzed the up-regulated genes of the treatment groups.
The Horcha-6-H group presented the most significant effect with 86 up-regulated genes (Figure 4E).We performed GO analysis on these 86 up-regulated genes and found that most genes were related to the signal transduction, hormone metabolism, and cytomembrane component (Figure 4F).The result of the KEGG enrichment analysis indicated that these 86 genes were significantly enriched in the cellular differentiation-related signaling pathway, including TGF-β, Hippo, Wnt, and signaling pathways regulating pluripotency in stem cells (Figure 4G).The generated heat map indicated the condition of these 86 genes expressed in each group (Figure 4H).This result demonstrates that Horcha-6 treated the migraine by reducing the oxidative stress and regulating the vascular function, hormone metabolism, and cellular regeneration and repair.

Discussion
Traditional Mongolian medicine, as one of the outstanding cultural heritages of the Mongolian nationality, is a summary of the Mongolian experience in fighting against diseases for a long time and gradually formed by absorbing the experience of traditional Chinese medicine and traditional Tibetan medicine.The TMM has its unique theoretical system and many diagnosis and treatment methods, including various classic prescriptions [33,34].Horcha-6 is one of the classic prescriptions commonly used for migraines in clinics.According to the theories of TMM, it reduces headache symptoms and discomfort by reducing the localized vasospasm, relaxing the heart rate, and balancing the effects of the three body elements (Hey, Xila, and Badagen) [11,33].Migraine symptoms were decreasing physical activity and movement [35].After nitroglycerin administration, we observed that rats exhibited abnormal behaviors similar to other studies.For example, exploring, locomoting, and shuttling are reduced, while freezing, resting, staying in a dark environment, and grooming is increased.[36].After administration with Horcha-6 some of these nitroglycerin-induced migraine symptoms have been alleviated.In the LC-MS/M assay and network analysis, we detected 55 bio-active components of Horcha-6, and 163 migraine-related targets of 55 components; then, through the GO and KEGG analysis, we found Wnt, TGF-β, Hippo, fluid shear stress, and vascular smooth muscle contraction signaling pathways.These signaling pathways are directly correlation with migraine.
The pathological investigation showed that inflammation, vascular diseases, and hormone dysregulation were involved in the development of migraines [37][38][39][40][41].Our RNA sequencing results identified 127 differentially expressed genes, including up-regulated and down-regulated genes.After GO and KEGG enrichment analyzing these identified genes, we found that Horcha-6 has significantly down-regulated fluid shear stress and vascular smooth muscle contraction signaling pathways related gens.Vascular smooth muscle contraction is triggered by increased intracellular free calcium concentration ([Ca 2+ ]i), promoting actin-myosin cross-bridge formation and reversal of vascular smooth muscle contraction-importance of vascular relaxation.Vascular smooth muscle relaxation occurs as due to decreased [Ca 2+ ]i.Vascular relaxation usually has an antihypertensive effect and, and antihypertensive medicine is used for migraine prevention [42,43].So, Horcha-6 may relieve migraines through the antihypertensive mechanism of vascular relaxation.In addition, the treatment of Horcha-6 has up-regulated the signaling pathways of Wnt, TGF-β, and Hippo pathways related genes.These results were consistent with previous finding and the results of network analysis.Previous studies show that Wnt activators that restore Wnt/β-catenin signalling in brain could represent therapeutic tools against migraine [44].Here, Horcha-6 may be through the Wnt signaling pathways to relieve migraine.
We also found 31 chemical components of Horch-6 related to those signaling pathways, in which quercetin, luteolin, and kaempferol played major roles in regulating those signaling pathways.Quercetin can downregulate the expressions of genes related to vascular smooth muscle contraction in vascular smooth muscle cells [45].Luteolin can upregulated the expressions of genes related to Wnt and Hippo signaling pathways in neural stem cells [46].Kaempferol can activate the Wnt signaling pathway [47].Therefore, Horch-6, through the active components to upregulated and downregulated genes for treating migraine, can be seen.

Conclusions
Horcha-6 can relieve migraines through its bio-active components quercetin, luteolin, and kaempferol to modulate fluid shear stress, vascular smooth muscle contraction, TGF-β, Hippo, and Wnt signaling pathways which directly related the initiation and development of migraine.However, the indeed mechanisms of these components how to fix up migraine are still needed more many studies to reveal.

Figure 1
Figure 1 Rat migraine models were established and the Horcha-6 effect on the rat behaviors was detected during the migraine interictal state.(A) A schematic diagram of the timeline of rat model construction and related assays.(B) Results of rat body weight measurements during the fifteen days.(C) The first test results of rat behaviors included exploring, locomotion, freezing, grooming, and light-aversive behavior, as well as spontaneous tactile allodynia.Treatment with Horcha-6 relieved the rat abnormal behaviors and pain.(D) The second test results of rat behaviors included exploring, locomotion, freezing, grooming, and light-aversive behavior, as well as spontaneous tactile allodynia.Treatment with Horcha-6 relieved the rat abnormal behaviors and pain.Data are compared between the control and model group ( * P < 0.05, ** P < 0.01), compared between the model and treatment groups ( # P < 0.05, ## P < 0.01).

Figure 2
Figure 2 An overview of the Horcha-6 components detected by liquid chromatography with tandem mass spectrometry assay.(A) The fingerprint of the Horcha-6 samples components detected in the negative electrospray ionization mode.(B) The enrichment analysis of Horcha-6 metabolites was detected in the negative electrospray ionization mode according to the HMDB annotation.(C) The KEGG pathway annotation of the Horcha-6 metabolites detected in the negative electrospray ionization mode.(D) The fingerprint of the Horcha-6 samples components detected in the positive electrospray ionization mode.(E) The enrichment analysis of the Horcha-6 metabolites detected in the positive electrospray ionization mode according to the HMDB annotation.(F) The KEGG pathway annotation of the Horcha-6 metabolites detected in the positive electrospray ionization mode.HMDB, Human Metabolome Database; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 4 RNA
Figure 4 RNA sequencing results indicated that Horcha-6 could regulate the mRNA levels in the trigeminal nerve of rat migraine models.(A) The Venn diagram shows the relationship of the down-regulated genes between the treatment groups.The down-regulatory effect of the Horcha-6-H group was the most significant, and 41 genes were significantly down-regulated (n = 3, P < 0.05).(B) The GO enrichment results of the Horcha-6-H group down-regulated genes (top 10 of pathway enrichment in each part of biological process; cellular components; molecular function).(C) In the KEGG enrichment results of the down-regulated genes in the Horcha-6-H group, the top 20 KEGG signaling pathways have been shown.(D) The heat map shows the mRNA levels of these 41 down-regulated genes by Horcha-6-H in each group sample.(E) The Venn diagram shows the relationship of up-regulated genes between the three treatment groups.The up-regulatory effect of the Horcha-6-H group was the most significant, and 86 genes were significantly up-regulated (n = 3, P < 0.05).(F) The GO enrichment results of the Horcha-6-H group up-regulated genes (top 10 of pathway enrichment in each part of biological process; cellular components; molecular function).(G) In the KEGG enrichment results of up-regulated genes in the Horcha-6-H group, the top 20 KEGG signaling pathways have been shown.(H) The heat map shows the mRNA levels of these 86 up-regulated genes by Horcha-6-H in each group sample.KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology.