Paeonol and glycyrrhizic acid in combination ameliorate the recurrent nitroglycerin-induced migraine-like phenotype in rats by regulating the GABBR2/TRPM8/PRKACA/TRPV1 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Paeonol (PAE) and glycyrrhizic acid (GLY) are predominate components of 14 blood-entering ones of Piantongtang No. 1, which is a traditional Chinese medicine prescription for chronic migraine with minimal side effects. Both paeonol and glycyrrhizic acid exhibit analgesic, neuroprotective and anti-inflammatory properties individually. Our previous research has highlighted their combined effect (PAE + GLY) in ameliorating migraine symptoms. However, there are not yet any studies exploring the mechanism of action of PAE + GLY in the treatment of migraine. AIM OF THE STUDY: This research aimed to determine the mechanism of PAE + GLY in ameliorating the recurrent nitroglycerin-induced migraine-like phenotype in rats. MATERIALS AND METHODS: Using a nitroglycerin-induced migraine model via subcutaneous injection in the neck, we evaluated the effect of PAE + GLY on migraine-like symptoms. Behavioural tests and biomarkers analysis were employed, alongside transcriptome sequencing (RNA-seq). Mechanistic insights were further verified utilising reverse transcription quantitative PCR (RT-qPCR), Western blot (WB), ELISA and immunofluorescence (IF) techniques. RESULTS: Following treatment with PAE + GLY, hyperalgesia threshold and 5-hydroxytryptamine (5-HT) levels increased, and migraine-like head scratching, histamine and calcitonin gene-related peptide (CGRP) levels were reduced. RNA-Seq experiments revealed that PAE + GLY upregulated the expression of Glutamate decarboxylase 2 (GAD2) and γ-aminobutyric acid type B receptor subunit 2 (GABBR2) genes. This upregulation activated the GABAergic synapse pathway, effectively inhibiting migraine attacks. Further validation demonstrated an increase in γ-aminobutyric acid (GABA) content in cerebrospinal fluid post PAE + GLY treatment, coupled with increased expression of dural GAD2, GABBR2 and transient receptor potential channel M8 (TRPM8). Consequently, this inhibited the expression of dural cAMP-dependent protein kinase catalytic subunit alpha (PRKACA) and transient receptor potential channel type 1 (TRPV1), subsequently downregulating p-ERK1/2, p-AKT1, IL-1ß and TNF-α. CONCLUSIONS: Our findings underscore that PAE + GLY ameliorates inflammatory hyperalgesia migraine by upregulating inhibitory neurotransmitters and modulating the GABBR2/TRPM8/PRKACA/TRPV1 pathway.

Screening and evaluation of quality markers of Radix Cudramiae for liver disease based on an integrated strategy of in vivo pharmacokinetics and in vitro HPLC fingerprint.

Radix Cudramiae, the dried root of Cudrania cochinchinensis (Lour.) Kudo et Masam., is a valuable ethnomedicine with outstanding antihepatitis activity. However, the lack of reports on quality markers (Q-markers) hindered its quality evaluation and standardization, as a result restricted its clinical application. This paper aimed to discover the Q-markers of Radix Cudramiae with a comprehensive strategy based on in vivo pharmacokinetics and in vitro HPLC fingerprint. A rapid and sensitive ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) analytical method was firstly developed and validated for simultaneous determination of six potential active ingredients (eriodictyol, dihydrokaempferol, dihydromorin, kaempferol, naringenin and morin) of Radix Cudramiae in rat plasma and tissues, which was successfully applied to the holistic comparison of pharmacokinetics and tissue distribution between normal and acute liver injury rats. On the other hand, a representative HPLC fingerprint of Radix Cudramiae was also established to elucidate the chemical profile for overall quality evaluation. Dihydrokaempferol-7-O-ß-D-glucoside (the naturally existed chemical formation of dihydrokaempferol) and kaempferol screened out with high exposure levels in vivo and high resolution in HPLC fingerprint were finally selected as Q-markers of Radix Cudramiae. To the best of our knowledge, it was the first time for people to discover in vivo properties and pharmacokinetic parameters of components in Radix Cudramiae, as well as the first report on its representative HPLC fingerprint. Also, the integrated strategy could offer an effective way for TCMs Q-markers screening.

Identification of key signaling pathways in cerebral small vessel disease using differential pathway network analysis.

Cerebral small vessel disease (CSVD) primarily affects the perforating cerebral arterioles and capillaries, and results in injury to subcortical grey and white matter. Despite advances in determining the genetic basis of CSVD, the molecular mechanisms underlying the development and progression of CSVD remain unclear. The present study aimed to identify significant signaling pathways associated with CSVD based on differential pathway network analysis. Combining CSVD microarray data with human protein-protein interaction data and data from the Reactome pathway database, pathway interactions were constructed using the Spearman's correlation coefficient strategy. Pathway interactions with weight values of >0.95 were selected to construct the differential pathway network, which contained 715 differential pathway interactions covering 312 nodes and was visualized using Cytoscape software. A total of 15 hub pathways with a top 5% degree distribution in the differential pathway network were identified. The top 5 hub pathways were associated with the synthesis and metabolism of fatty acids. The results of the present study indicate that the synthesis and metabolism of fatty acids is associated with the occurrence and development of CSVD, and may thus provide insights to improve the early diagnosis and treatment of CSVD.

Effects of Zusanli and Ashi Acupoint Electroacupuncture on Repair of Skeletal Muscle and Neuromuscular Junction in a Rabbit Gastrocnemius Contusion Model.

Objective. To explore the effects of electroacupuncture (EA) at ST36 (EA-ST36) and at Ashi acupoints (EA-Ashi) on skeletal muscle repair. Methods. Seventy-five rabbits were randomly divided into five groups: normal, contusion, EA-Ashi, EA-ST36, and EA at Ashi acupoints and ST36 (EA-AS). EA (0.4 mA, 2 Hz, 15 min) was applied after an acute gastrocnemius contusion. The morphology of myofibers and neuromuscular junctions (NMJs) and expressions of growth differentiation factor-8 (GDF-8), acetylcholinesterase (AChE), Neuregulin 1 (NGR1), and muscle-specific kinase (MuSK) were assessed 7, 14, and 28 days after contusion. Results. Compared with that in contusion group, there was an increase in the following respective parameters in treatment groups: the number and diameter of myofibers, the mean staining area, and continuities of NMJs. A comparison of EA-Ashi and EA-ST36 groups indicated that average myofiber diameter, mean staining area of NMJs, and expressions of AChE and NRG1 were higher in EA-Ashi group, whereas expression of GDF-8 decreased on day 7. However, increases in myofiber numbers, expressions of MuSK and AChE, as well as decreases in GDF-8 expression, and the discontinuities were observed in EA-ST36 group on the 28th day. Conclusion. Both EA-ST36 and EA-Ashi promoted myofiber regeneration and restoration of NMJs. EA-Ashi was more effective at earlier stages, whereas EA-ST36 played a more important role at later stages.

Effects of electroacupuncture on recovery of the electrophysiological properties of the rabbit gastrocnemius after contusion: an in vivo animal study.

BACKGROUND: Our preliminary studies indicated that electroacupuncture (EA) at the ST36 and Ashi acupoints could promote regeneration of the rabbit gastrocnemius (GM) by improving microcirculation perfusion, promoting the recovery of myofiber structures, and inhibiting excessive fibrosis. However, the effects of EA on recovery of the electrophysiological properties of the GM after contusion are not yet clear. Thus, the purpose of this study was to investigate the effects of EA at the Zusanli (ST36) and Ashi acupoints with regard to recovery of the electrophysiological properties of the rabbit GM after contusion. METHODS: Forty-five rabbits were randomly divided into three groups: normal, contusion, and EA. After an acute GM contusion was produced (in rabbits in the contusion and EA groups), rabbits in the EA group were treated with electrostimulation at the ST36 and Ashi acupoints with 0.4 mA (2 Hz) for 15 min. The contusion group received no EA treatment. At different time points (7, 14, and 28 days) after contusion, we performed surface electromyography (EMG) and measured the nerve conduction velocity (NCV) of the GM and the GM branch of the tibial nerve. We also examined acetylcholinesterase (AchE) and Agrin expression in the neuromuscular junction (NMJ) via immunohistochemistry. RESULTS: Compared with the contusion group, the EMG amplitude and NCV in rabbits in the EA group were significantly higher at all time points after contusion. AchE and Agrin expression in the EA group were significantly higher than those in the contusion group. CONCLUSIONS: Our results showed that EA at the ST36 and Ashi acupoints effectively promoted recovery of the electrophysiological properties of the rabbit GM after contusion. The effects of EA were realized by promotion of the regeneration of myofibers and nerve fibers, as well as acceleration of NMJ reconstruction by upregulation of AchE and Agrin expression in the motor endplate area.

The time course effects of electroacupuncture on promoting skeletal muscle regeneration and inhibiting excessive fibrosis after contusion in rabbits.

The aim of this study was to investigate the longitudinal effects of electroacupuncture (EA) on Zusanli (ST36) and Ashi acupoints in promoting skeletal muscle regeneration and inhibiting excessive fibrosis after contusion in rabbits. Sixty rabbits were randomly divided into four groups: normal, contusion, EA, and recombinant human insulin-like growth factor-I (rhIGF-I). An acute skeletal muscle contusion was produced on the right gastrocnemius (GM) by an instrument-based drop-mass technique. EA was performed for 15 minutes every two days with 0.4 mA (2 Hz), and GM injections were executed with rhIGF-I (0.25 mL once a week). Rabbits treated with EA had a higher T-SOD and T-AOC serum activities and lower MDA serum level, the blood perfusion of which was also significantly higher. In the EA group, the diameter of the myofibril was uniform and the arrangement was regular, contrary to the contusion group. The number and diameter of regenerative myofibers and MHC expression were increased in the EA group. EA treatment significantly decreased fibrosis formation and reduced both GDF-8 and p-Smad2/3 expressions in injured muscle. Our data indicate that EA may promote myofiber regeneration and reduce excessive fibrosis by improving blood flow and antioxidant capacities. Additionally, EA may regulate signaling factor expression after contusion.