Proteomics profiling reveals mitochondrial damage in the thalamus in a mouse model of chronic migraine.

BACKGROUND: Migraine, a complex brain disorder, is regarded as a possible clinical manifestation of brain energy dysfunction. The trigeminovascular system is considered the basis for the pathogenesis of migraine, hence we depicted the proteomics profiling of key regions in this system, then focusing on protein alterations related to mitochondrial function. The aim of this study is to illustrate the role of mitochondria in migraine. METHODS: A mouse model of chronic migraine (CM) was established by repeated nitroglycerin (NTG) stimulation and evaluated by von-Frey filaments, a hot plate and a light-dark box. Differentially expressed proteins (DEPs) in some subcortical brain regions of the trigeminovascular system were screened through liquid chromatography-tandem mass spectrometry (LC‒MS/MS) to analyse the specificity of key signaling pathways in different brain regions. And then mitochondrial function, structure and dynamics were determined by qPCR, ELISA, and transmission electron microscope (TEM). Finally, the effect of mitochondrial intervention-Urolithin A (UA) on CM was investigated. RESULTS: Repeated NTG injection triggered photophobia, periorbital and hind paw allodynia in mice. The proteomics profiling of CM model showed that 529, 109, 163, 152 and 419 DEPs were identified in the thalamus, hypothalamus, periaqueductal grey (PAG), trigeminal ganglion (TG) and trigeminocervical complex (TCC), respectively. The most significant changes in the brain region-specific pathways pointed to thalamic mitochondrial impairment. NTG induced mitochondrial structural disruption, dysfunction and homeostatic dysregulation, which could be partially attenuated by UA intervention. CONCLUSION: Our findings highlight the involvement of mitochondrial damage in the thalamus in central sensitization of CM, which provides evidence of possible metabolic mechanisms in migraine pathophysiology.

Tyrosine hydroxylase involved in cuticle tanning and reproduction in the 28-spotted potato ladybeetle, Henosepilachna vigintioctopunctata.

BACKGROUND: Tyrosine hydroxylase (TH), a melanin synthesis pathway enzyme hydroxylating tyrosine into 3,4-dihydroxyphenylalanine, is involved in the pigmentation and sclerotization of insect cuticles. However, the role of TH in 28-spotted potato ladybeetle (Henosepilachna vigintioctopunctata), an emerging pest of the solanaceous crops has been explored to a limited extent. In this study, we integrated dietary RNA interference (RNAi) and hematoxylin and eosin (H&E) staining with various bioassays to analyze the role of tyrosine hydroxylase (HvTH) throughout the developmental processes of Henosepilachna vigintioctopunctata. RESULTS: The results revealed that ingestion of dsHvTH led to cuticle tanning impairment, arrested larval feeding in the first and second instars of Henosepilachna vigintioctopunctata, and subsequently resulted in 100% mortality. The H&E staining assays revealed that dsHvTH prevented new abdominal cuticle formation. A pharmacological study using 3-iodo-tyrosine (3-IT), a HvTH inhibitor, disrupted larval-larval-pupal cuticle tanning during the third-fourth instar larval development and eventually failed to pupate. Similarly, dsHvTH fed to fourth instars hindered larval-pupal-adult cuticle tanning, and the eclose adults were 100% malformed. Ingestion of dsHvTH or 3-IT significantly down-regulated HvTH, HvDDC, Hvebony, and Hvlaccase2 expression and reduced dopamine levels. Finally, HvTH silencing in adult females substantially reduced the offspring hatching rates. CONCLUSIONS: The collective results of the study suggested that HvTH plays conserved roles in larval-pupal-adult cuticle melanization and sclerotization while exhibiting a novel function in Henosepilachna vigintioctopunctata reproduction. © 2022 Society of Chemical Industry.

RNAi suppression of the nuclear receptor FTZ-F1 impaired ecdysis, pupation, and reproduction in the 28-spotted potato ladybeetle, Henosepilachna vigintioctopunctata.

Fushi-tarazu factor 1 (FTZF1) is an ecdysone-inducible transcription factor that plays a vital role during the metamorphosis in insects. In this study, we functionally characterized HvFTZ-F1 in H. vigintioctopunctata, a dreadful solanaceous crop pest, by using a dietary RNA interference technique. The HvFTZ-F1 expression levels were elevated in the 1st and 2nd-instars before molting and declined immediately after ecdysis. The HvFTZ-F1 silencing led to high mortality in the 1st instars, while the expression of the osmosis-regulative gene, HvAQPAn.G, was significantly increased in the 1st instars. HvFTZ-F1 silencing downregulated the Halloween and 20E-related genes, decreased the ecdysteroids titer, suppressed the expression of pigmentation-related genes, and reduced the catecholamines titer. In the 4th instars, HvFTZ-F1 silencing caused 100% mortality by arresting the development at the prepupal stage and preventing new abdominal cuticle formation. In the female adults, HvFTZ-F1 silencing caused an evident decrease in fecundity, prolonged the pre-oviposition period, reduced the number of eggs and hatching rate, severely atrophied the ovaries. Moreover, the 20E-related genes and the dopamine synthesis genes were suppressed in the dsHvFTZ-F1-treated females. Overall, our results revealed that HvFTZ-F1 regulates ecdysis, pupation, and reproduction in H. vigintioctopunctata, thereby could be a promising molecular target for the development of RNAi-based biopesticides to control H. vigintioctopunctata.

Oral RNAi toxicity assay suggests clathrin heavy chain as a promising molecular target for controlling the 28-spotted potato ladybird, Henosepilachna vigintioctopunctata.

BACKGROUND: Use of RNA interference (RNAi) technology in effective pest management has been explored for decades. Henosepilachna vigintioctopunctata is a major solanaceous crop pest in Asia. In this study, the effects of the RNAi-mediated silencing of clathrin heavy chain in H. vigintioctopunctata were investigated. RESULTS: Feeding either the in vitro-synthesized or the bacterially expressed double-stranded RNAs (dsRNAs) significantly impaired the normal physiology of H. vigintioctopunctata instars and adults. However, the bacterially expressed dsHvChc caused higher mortality than the in vitro-synthesized ones in the larvae and adults. Moreover, on evaluating the potential risk of dsHvChc on Propylea japonica, significant transcriptional effects of dsHvChc1 were observed, while the organismal level effects were not significant. On the contrary, dsHvChc2 did not affect P. japonica at either level. A similar test revealed significant transcriptional effects of dsPjChc1 on H. vigintioctopunctata, while staying ineffective at the organismal levels. Conversely, dsPjChc2 did not affect H. vigintioctopunctata at either level. Importantly, no effect of dsPjChc1 exposure on H. vigintioctopunctata suggested that other factors besides the 21-nucleotide (nt) matches between sequences were responsible. Finally, ingestion of dsHvmChc1 derived from H. vigintioctomaculata, containing 265-nt matches with dsHvChc1, caused 100% mortality in H. vigintioctopunctata. CONCLUSIONS: We conclude that (i) species with numerous 21-nt matches in homologous genes are more likely to be susceptible to dsRNA; (ii) dsRNA can be safely designed to avoid negative effects on non-target organisms at both transcriptional and organismal levels; (iii) HvChc can be used as an efficient RNAi target gene to effectively manage H. vigintioctopunctata. © 2021 Society of Chemical Industry.

Inhibitory effects of quercetin and its major metabolite quercetin-3-O-ß-D-glucoside on human UDP-glucuronosyltransferase 1A isoforms by liquid chromatography-tandem mass spectrometry.

Quercetin is a flavonoid that is widely present in plant-derived food. Quercetin-3-O-ß-D-glucoside (Q3GA) is a predominant metabolite of quercetin in animal and human plasma. The inhibitory effects of the UDP-glucuronosyl transferases (UGTs) caused by herbal components may be a key factor for the clinical assessment of herb-drug interactions (HDIs). The present study aimed to investigate the inhibitory profile of quercetin and Q3GA on recombinant UGT1A isoforms in vitro. The metabolism of the nonspecific substrate 4-methylumbelliferone (4-MU) by the UGT1A isoforms was assessed by liquid chromatography-tandem mass spectrometry. Preliminary screening experiments indicated that quercetin exhibited stronger inhibitory effects on UGT1A1, UGT1A3, UGT1A6 and UGT1A9 enzymes than Q3GA. Kinetic experiments were performed to characterize the type of inhibition caused by quercetin and Q3GA towards these UGT isoforms. Quercetin exerted non-competitive inhibition on UGT1A1 and UGT1A6, with half maximal inhibitory concentration (IC50) values of 7.47 and 7.07 µM and inhibition kinetic parameter (Ki) values of 2.18 and 28.87 µM, respectively. Quercetin also exhibited competitive inhibition on UGT1A3 and UGT1A9, with IC50 values of 10.58 and 2.81 µM and Ki values of 1.60 and 0.51 µM, respectively. However, Q3GA displayed weak inhibition on UGT1A1, UGT1A3 and UGT1A6 enzymes with IC50 values of 45.21, 106.5 and 51.37 µM, respectively. In the present study, quercetin was a moderate inhibitor of UGT1A1 and UGT1A3, a weak inhibitor of UGT1A6, and a strong inhibitor on UGT1A9. The results of the present study suggested potential HDIs that may occur following quercetin co-administration with drugs that are mainly metabolized by UGT1A1, UGT1A3 and UGT1A9 enzymes.

Inhibition effect of epigallocatechin-3-gallate on the pharmacokinetics of calcineurin inhibitors, tacrolimus, and cyclosporine A, in rats.

BACKGROUND: Epigallocatechin-3-gallate (EGCG) is the most biologically active catechin of green tea. Tacrolimus (TAC) and cyclosporine A (CsA) are immunosuppressive agents commonly used in clinical organ transplantation. The present study investigated the effect of EGCG on the pharmacokinetics of TAC and CsA in rats and its underlying mechanisms. RESEARCH DESIGN AND METHODS: Either TAC or CsA was administered to rats intravenously or orally with or without concomitant EGCG. Polymerase Chain Reaction and Western Blot were used to determine the effect of EGCG on drug-metabolizing enzymes (DMEs), drug transporters (DTs) and nuclear receptors (NRs). RESULTS: The Cmax and AUC of TAC were reduced, and V/F and CL/F of TAC were enhanced after co-administration of EGCG. EGCG increased the Cmax, AUC of CsA at 3 ~ 30 mg∙kg-1 dosages, while decreased those parameters at the dosage of 100 mg∙kg-1. EGCG inhibited the mRNA and protein expressions of DMEs and DTs, such as CYP3A1, A2, UGT1A1, Mdr1 and Mrp2, but upregulated the expressions of Car, Pxr and Fxr. CONCLUSIONS: These results revealed consumption of high dose EGCG may cause a significant alteration in pharmacokinetics of TAC and distribution/elimination profiles of CsA through the regulation of DMEs, DTs and NRs.

Glycyrrhizic Acid Ameliorates Cognitive Impairment in a Rat Model of Vascular Dementia Associated with Oxidative Damage and Inhibition of Voltage-Gated Sodium Channels.

Vascular dementia (VD) is the second most common cause of cognitive impairment in the elderly population. Our study aims to investigate the neuroprotective effects of glycyrrhizic acid (GA), a major active constituent of Glycyrrhiza glabra root, in a VD rat model induced by permanent occlusion of the bilateral common carotid arteries. Spatial cognitive function was examined by the Morris water maze test and synaptic plasticity was explored by assessing long-term potentiation. The results showed that GA (20 mg/kg for 5 days) significantly improved the performance of learning and memory of VD rats in the Morris water maze test and attenuated induction of long-term potentiation. Histopathological studies showed that GA significantly attenuated cell damage in VD rats. Malondialdehyde levels and superoxide dismutase activity were analyzed in the hippocampus and cortex to investigate anti-oxidant status. The results showed that GA decreased the level of lipid peroxidation and increased the activity of superoxide dismutase in VD rats. Lastly, whole-cell patch-clamp analysis was used to examine the effect of GA on voltage-gated sodium channels (VGSCs) in hippocampal CA1 pyramidal neurons. GA (10, 20 and 50 µM) inhibited the current amplitude of the VGSCs. These results suggest that the neuroprotective e.ects of GA in VD rats relate to the reduction of oxidative stress and inhibition of VGSCs. Our study provides experimental evidence for the application of GA in the treatment of cognitive deficits induced by Alzheimer's disease, stroke, or traumatic brain injury.

Cholecystokinin-8 antagonizes electroacupuncture analgesia through its B receptor in the caudate nucleus.

OBJECTIVES: The analgesic effect of electroacupuncture (EA) stimulation has been proved. However, its mechanism of action is not clear. It has been well-known that cholecystokinin-8 (CCK-8) is a neuropeptide which is mainly related to the mediation of pain. The caudate nucleus was selected to determine if the release of CCK and the neural activity in this nucleus were involved in producing EA analgesia. MATERIALS AND METHODS: Radiant heat focused on the rat-tail was used as the noxious stimulus. The pain threshold of rats was measured by tail-flick latency (TFL). EA stimulation at the bilateral Zusanli (ST 36) acupoints of rats was used to investigate the effects of EA analgesia. The electrical activities of pain-excited neurons (PEN) and pain-inhibited neurons (PIN) in the caudate nucleus were recorded with a glass microelectrode. The present study examined the antagonistic effects of the intracerebral ventricular injection of CCK-8 on EA analgesia and reversing effects of CCK-B receptor antagonist (L-365,260) injection into the caudate nucleus on CCK-8. RESULTS: The radiant heat focused on the tail of rats caused an increase in the evoked discharge of PEN and a reduction in the evoked discharge of PIN. EA stimulation at the bilateral ST 36 acupoints of rats resulted in the inhibition of PEN, the potentiation of PIN, and prolongation of TFL. The analgesic effect of EA was antagonized when CCK-8 was injected into the intracerebral ventricle of rats. The antagonistic effect of CCK-8 on EA analgesia was reversed by injection of CCK-B receptor antagonist (L-365,260) into the caudate nucleus of rats. CONCLUSIONS: Our results suggest that CCK-8 antagonize EA analgesia through its B receptor.