Anterior cingulate cortex-related functional hyperconnectivity underlies sensory hypersensitivity in Grin2b-mutant mice.

Sensory abnormalities are observed in ~90% of individuals with autism spectrum disorders (ASD), but the underlying mechanisms are poorly understood. GluN2B, an NMDA receptor subunit that regulates long-term depression and circuit refinement during brain development, has been strongly implicated in ASD, but whether GRIN2B mutations lead to sensory abnormalities remains unclear. Here, we report that Grin2b-mutant mice show behavioral sensory hypersensitivity and brain hyperconnectivity associated with the anterior cingulate cortex (ACC). Grin2b-mutant mice with a patient-derived C456Y mutation (Grin2bC456Y/+) show sensory hypersensitivity to mechanical, thermal, and electrical stimuli through supraspinal mechanisms. c-fos and functional magnetic resonance imaging indicate that the ACC is hyperactive and hyperconnected with other brain regions under baseline and stimulation conditions. ACC pyramidal neurons show increased excitatory synaptic transmission. Chemogenetic inhibition of ACC pyramidal neurons normalizes ACC hyperconnectivity and sensory hypersensitivity. These results suggest that GluN2B critically regulates ASD-related cortical connectivity and sensory brain functions.

Effect of a differential training paradigm with varying frequencies and amplitudes on adaptation of vestibulo-ocular reflex in mice.

The vestibulo-ocular reflex (VOR) functions to maintain eye stability during head movement, and VOR gain can be dynamically increased or decreased by gain-up or gain-down adaptation. In this study, we investigated the impact of a differential training paradigm with varying frequencies and amplitudes on the level of VOR adaptation in mice. Training for gain-up (out of phase) or gain-down (in phase) VOR adaptation was applied for 60 min using two protocols: (1) oscillation of a drum and turntable with fixed frequency and differing amplitudes (0.5 Hz/2.5°, 0.5 Hz/5° and 0.5 Hz/10°). (2) Oscillation of a drum and turntable with fixed amplitude and a differing frequency (0.25 Hz/5°, 0.5 Hz/5° and 1 Hz/5°). VOR adaptation occurred distinctively in gain-up and gain-down learning. In gain-up VOR adaptation, the learned increase in VOR gain was greatest when trained with the same frequency and amplitude as the test stimulation, and VOR gain decreased after gain-up training with too high a frequency or amplitude. In gain-down VOR adaptation, the decrease in VOR gain increased as the training frequency or amplitude increased. These results suggest that different mechanisms are, at least in part, involved in gain-up and gain-down VOR adaptation.

Adaptation of Optokinetic Reflex by Training with Different Frequency and Amplitude.

BACKGROUND: Although the occurrence of optokinetic reflex (OKR) adaptation after OKR training is well established, the dynamic properties of OKR adaptation has not been fully studied. This study aimed to examine the difference in the amount of OKR adaptation according to OKR training protocols which have different frequency or amplitude of drum oscillation. METHODS: Using C57BL/6N male mice, we induced OKR adaptation by 3 different categories of learning paradigm as follows: (1) Optokinetic drum oscillation for 60 min with same amplitude and different frequency. (2) Optokinetic drum oscillation for 60 min with same frequency and different amplitude. (3) Training with serial combination of different frequency or amplitude. RESULTS: The results show that the amount of OKR adaptation was greater after OKR training with lower frequency or amplitude than that with higher frequency or amplitude. CONCLUSIONS: This finding may suggest that the retinal slip signal with lower-velocity OKR stimulation serves as more precise instructive signal for learning, leading to induction of more efficient training effect. Another interesting finding was that the OKR gain increase tended to be greater after training composed of sequential combination of decreasing frequency or amplitude than that composed of sequential combination of increasing frequency or amplitude. Furthermore, the OKR training with high frequency or amplitude eliminated a part of learning effects which have already formed by previous training. We postulate that the stimulation during training with high frequency or amplitude may implement a disturbing instruction for OKR learning when it is conducted in mice with increased OKR gain after previous OKR training.

Quantitative Proteomics Reveals Distinct Molecular Signatures of Different Cerebellum-Dependent Learning Paradigms.

The cerebellum improves motor performance by adjusting motor gain appropriately. As de novo protein synthesis is essential for the formation and retention of memories, we hypothesized that motor learning in the opposite direction would induce a distinct pattern of protein expression in the cerebellum. We conducted quantitative proteomic profiling to compare the level of protein expression in the cerebellum at 1 and 24 h after training from mice that underwent different paradigms of cerebellum-dependent oculomotor learning from specific directional changes in motor gain. We quantified a total of 43 proteins that were significantly regulated in each of the three learning paradigms in the cerebellum at 1 and 24 h after learning. In addition, functional enrichment analysis identified protein groups that were differentially enriched or depleted in the cerebellum at 24 h after the three oculomotor learnings, suggesting that distinct biological pathways may be engaged in the formation of three oculomotor memories. Weighted correlation network analysis discovered groups of proteins significantly correlated with oculomotor memory. Finally, four proteins (Snca, Sncb, Cttn, and Stmn1) from the protein group correlated with the learning amount after oculomotor training were validated by Western blot. This study provides a comprehensive and unbiased list of proteins related to three cerebellum-dependent motor learning paradigms, suggesting the distinct nature of protein expression in the cerebellum for each learning paradigm. The proteomics data have been deposited to the ProteomeXchange Consortium with identifiers .

STIM1 Regulates Somatic Ca2+ Signals and Intrinsic Firing Properties of Cerebellar Purkinje Neurons.

Control of Ca2+ flux between the cytosol and intracellular Ca2+ stores is essential for maintaining normal cellular function. It has been well established in both neuronal and non-neuronal cells that stromal interaction molecule 1 (STIM1) initiates and regulates refilling Ca2+ into the ER. Here, we describe a novel, additional role for STIM1, the regulation of free cytosolic Ca2+, and the consequent control of spike firing in neurons. Among central neurons, cerebellar Purkinje neurons express the highest level of STIM1, and they fire continuously in the absence of stimulation, making somatic Ca2+ homeostasis of particular importance. By using Purkinje neuron-specific STIM1 knock-out (STIM1PKO) male mice, we found that the deletion of STIM1 delayed clearance of cytosolic Ca2+ in the soma during ongoing neuronal firing. Deletion of STIM1 also reduced the Purkinje neuronal excitability and impaired intrinsic plasticity without affecting long-term synaptic plasticity. In vestibulo-ocular reflex learning, STIM1PKO male mice showed severe deficits in memory consolidation, whereas they were normal in memory acquisition. Our results suggest that STIM1 is critically involved in the regulation of the neuronal excitability and the intrinsic plasticity of the Purkinje neurons as well as cerebellar memory consolidation.SIGNIFICANCE STATEMENT Stromal interaction molecule 1 (STIM1), which regulates the refilling of ER Ca2+, has been investigated in several systems including the CNS. In addition to a previous study showing that STIM1 regulates dendritic ER Ca2+ refilling and mGluR1-mediated synaptic transmission, we provide compelling evidence describing a novel role of STIM1 in spike firing Purkinje neurons. We found that STIM1 regulates cytosolic Ca2+ clearance of the soma during spike firing, and the interruption of this cytosolic Ca2+ clearing disrupts neuronal excitability and cerebellar memory consolidation. Our results provide new insights into neuronal functions of STIM1 from single neuronal Ca2+ dynamics to behavior level.

Long-Term Depression of Intrinsic Excitability Accompanied by Synaptic Depression in Cerebellar Purkinje Cells.

Long-term depression (LTD) at the parallel fiber (PF)-to-cerebellar Purkinje cell (PC) synapse is implicated in the output of PCs, the sole output of the cerebellar cortex. In addition to synaptic plasticity, intrinsic excitability is also one of the components that determines PC output. Although long-term potentiation of intrinsic excitability (LTP-IE) has been suggested, it has yet to be investigated how PF-PC LTD modifies intrinsic excitability of PCs. Here, we show that pairing of the PF and climbing fiber (CF) for PF-PC LTD induction evokes LTD-IE in cerebellar PCs from male C57BL/6 mice. Interestingly, this intrinsic plasticity showed different kinetics from synaptic plasticity, but both forms of plasticity share Ca2+ signaling and protein kinase C pathway as their underlying mechanism. Although small-conductance Ca2+-activated K+ channels play important roles in LTP-IE, no direct implication has been found. After PF-PC LTD induction, neither the temporal summation of dendritic EPSP nor the power of spike frequency adaptation is changed, indicating that cerebellar LTD executes the information processing in a quantitative way without quality changes of synaptic integration and generation of output signals. Our results suggest that LTD-IE may have a synergistic effect with synaptic depression on the total net output of neurons by amplifying the modification of PF synaptic transmission.SIGNIFICANCE STATEMENT Although the output of Purkinje cells (PCs) is a critical component of cerebellum-dependent learning and memory, the changes of PC excitability when synaptic LTD occurs are unclear. Here, we show that the induction of PF-PC LTD evokes LTD-IE in PCs. Our observation complements previous intrinsic plasticity phenomenon of long-term potentiation of intrinsic excitability (LTP-IE), providing evidence for the idea that intrinsic plasticity has bidirectionality as synaptic plasticity. LTD-IE occurs together with synaptic LTD and both phenomena are dependent on the Ca2+ signaling pathway. Furthermore, our findings raise the prospect that this synaptic and intrinsic plasticity acts synergistically in PCs to modify neuronal activity in the same direction when learning occurs.

Lateralization of horizontal semicircular canal canalolithiasis and cupulopathy using bow and lean test and head-roll test.

Accurate lateralization is important to improve treatment outcomes in horizontal semicircular canal (HSCC) benign paroxysmal positional vertigo (BPPV). To determine the involved side in HSCC-BPPV, the intensity of nystagmus has been compared in a head-roll test (HRT) and the direction of nystagmus was evaluated in a bow and lean test (BLT). The aim of this study is to compare the results of a BLT with those of a HRT for lateralization of HSCC-canalolithiasis and cupulopathy (heavy cupula and light cupula), and evaluate treatment outcomes in patients with HSCC-canalolithiasis. We conducted retrospective case reviews in 66 patients with HSCC-canalolithiasis and 63 patients with HSCC-cupulopathy. The affected side was identified as the direction of bowing nystagmus on BLT in 55 % (36 of 66) of patients with canalolithiasis, which was concordant with the HRT result in 67 % (24 of 36) of cases (concordant group). Lateralization was determined by comparison of nystagmus intensity during HRT in 30 patients who did not show bowing or leaning nystagmus. The remission rate after the first treatment was 71 % (17 of 24) in the concordant group and 45 % (5 of 11) in the discordant group. Both bowing and leaning nystagmus were observed in all patients with cupulopathy, and the side of the null plane was identified as the affected side. In conclusion, bowing and/or leaning nystagmus were observed in only 55 % of patients with HSCC-canalolithiasis, and the first treatment based on the result of BLT alone was effective in only 45 % of the patients in whom the BLT and HRT were discordant, which may suggest that the usefulness of BLT in lateralizing the HSCC-canalolithiasis may be limited.

Adaptation of vestibulo-ocular and optokinetic reflexes after massed and spaced vestibulo-ocular motor learning.

Although the superiority of spaced training over massed training has been established in many forms of learning, the learning efficacy between the two with respect to time efficiency may not be simply compared because a longer total duration of learning is required in spaced training than massed training due to spacing intervals intervening between training sessions in the former. The purpose of the present study was to evaluate the differences in the adaptation of the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) after visuo-vestibular training, and to investigate the efficacy of spaced and massed training in mice. Associative visuo-vestibular stimulation was applied to induce VOR and OKR motor learning. Training paradigms were categorized into five groups according to the duration of the spacing interval, keeping the total training time including spacing equal in all training paradigms. Both gain-up VOR training, which increased VOR gain and gain-down VOR training, which decreased VOR gain, increased OKR gain in the massed and spaced learning paradigms. While the increment in OKR gain after gain-up and gain-down training was maintained at 48 h after the end of the last training session, the change in VOR gain by gain-up or gain-down training recovered gradually after training. The OKR adaptation was still in progress during the spacing interval, and the amount of gain increase was greater with longer spacing interval. On the other hand, the VOR gain change after gain-up and gain-down training substantially recovered during the spacing interval. In conclusion, the present study, using learning paradigms with same total duration of training, demonstrated that the spacing effect was more robust in the adaptation of OKR than that of VOR, and the learning effect was maintained longer in OKR than in VOR. These differences in the adaptation of VOR and OKR following identical training conditions suggest that multiple plasticity mechanisms may be differentially involved in the gaze stabilization circuitry.

Minhee Analysis Package: an integrated software package for detection and management of spontaneous synaptic events.

To understand the information encoded in a connection between the neurons, postsynaptic current (PSC) has been widely measured as a primary index of synaptic strength in the field of neurophysiology. Although several automatic detection methods for PSCs have been proposed to simplify a workflow in the analysis, repetitive steps such as quantification and management of PSC data should be still performed with much effort. Here, we present Minhee Analysis Package, an integrated standalone software package that is capable of detecting, sorting, and quantifying PSC data. First, we developed a stepwise exploratory algorithm to detect PSC and validated our detection algorithm using the simulated and experimental data. We also described all the features and examples of the package so that users can use and follow them properly. In conclusion, our software package is expected to improve the convenience and efficiency of neurophysiologists to analyze PSC data by simplifying the workflow from detection to quantification. Minhee Analysis Package is freely available to download from http://www.github.com/parkgilbong/Minhee_Analysis_Pack .

Efficacy of spaced learning in adaptation of optokinetic response.

INTRODUCTION: The superiority of spaced training, in which repeated training sessions are given with resting intervals, over massed training in learning efficacy has been well established. However, longer duration of total training time has been required for spaced training than massed training because spacing intervals intervene between training sessions in spaced training. Thus, the learning efficacy may not be simply compared between spaced and massed training in terms of "time efficiency." The aim of the present study was to investigate the efficacy of spaced and massed training using adaptation of horizontal optokinetic reflex (hOKR) in mice. METHODS: Training paradigms were categorized into seven groups according to the duration of spacing interval, keeping total duration of hOKR training including spacing almost equal in all training paradigms. RESULTS: The amount of short-term hOKR gain increase immediately after the 60 min hOKR training was not significantly different among seven training paradigms. The hOKR adaptation was still in progress during a spacing interval, and the increment in hOKR gain tended to be greater with the longer spacing interval. The increase in hOKR gain was maintained until 48 hr after the end of training in both massed and spaced training. CONCLUSION: The short-term learning effect was not significantly different among training paradigms regardless of spacing interval in hOKR adaptation, which suggests that the spacing effect is robust enough to overcome the shortage of optokinetic training cycles in hOKR adaptation.

Kdm3b haploinsufficiency impairs the consolidation of cerebellum-dependent motor memory in mice.

Histone modifications are a key mechanism underlying the epigenetic regulation of gene expression, which is critically involved in the consolidation of multiple forms of memory. However, the roles of histone modifications in cerebellum-dependent motor learning and memory are not well understood. To test whether changes in histone methylation are involved in cerebellar learning, we used heterozygous Kdm3b knockout (Kdm3b+/-) mice, which show reduced lysine 9 on histone 3 (H3K9) demethylase activity. H3K9 di-methylation is significantly increased selectively in the granule cell layer of the cerebellum of Kdm3b+/- mice. In the cerebellum-dependent optokinetic response (OKR) learning, Kdm3b+/- mice show deficits in memory consolidation, whereas they are normal in basal oculomotor performance and OKR acquisition. In addition, RNA-seq analyses revealed that the expression levels of several plasticity-related genes were altered in the mutant cerebellum. Our study suggests that active regulation of histone methylation is critical for the consolidation of cerebellar motor memory.

Ablation of STAT3 in Purkinje cells reorganizes cerebellar synaptic plasticity in long-term fear memory network.

Emotional memory processing engages a large neuronal network of brain regions including the cerebellum. However, the molecular and cellular mechanisms of the cerebellar cortex modulating the fear memory network are unclear. Here, we illustrate that synaptic signaling in cerebellar Purkinje cells (PCs) via STAT3 regulates long-term fear memory. Transcriptome analyses revealed that PC-specific STAT3 knockout (STAT3PKO) results in transcriptional changes that lead to an increase in the expression of glutamate receptors. The amplitude of AMPA receptor-mediated excitatory postsynaptic currents at parallel fiber (PF) to PC synapses was larger in STAT3PKO mice than in wild-type (WT) littermates. Fear conditioning induced long-term depression of PF-PC synapses in STAT3PKO mice while the same manipulation induced long-term potentiation in WT littermates. STAT3PKO mice showed an aberrantly enhanced long-term fear memory. Neuronal activity in fear-related regions increased in fear-conditioned STAT3PKO mice. Our data suggest that STAT3-dependent molecular regulation in PCs is indispensable for proper expression of fear memory.

Decreased intrinsic excitability of cerebellar Purkinje cells following optokinetic learning in mice.

The optokinetic response (OKR), a reflexive eye movement evoked by a motion of the visual field, is known to adapt its strength to cope with an environmental change throughout life, which is a type of cerebellum-dependent learning. Previous studies suggested that OKR learning induces changes in in-vivo spiking activity and synaptic transmission of the cerebellar Purkinje cell (PC). Despite the recent emphasis on the importance of the intrinsic excitability related to learning and memory, the direct correlation between the intrinsic excitability of PCs and OKR learning has not been tested. In the present study, by utilizing the whole-cell patch-clamp recording, we compared the responses of cerebellar PCs to somatic current injection between the control and learned groups. We found that the neurons from the learned group showed a significant reduction in mean firing rate compared with neurons in the control group. In the analysis of single action potential (AP), we revealed that the rheobase current for the generation of single AP was increased by OKR learning, while AP threshold, AP amplitude, and afterhyperpolarization amplitude were not altered. Taken together, our result suggests that the decrease in the intrinsic excitability was induced in the cerebellar PC of learned group by an increase in the current threshold for generating AP.

Differential effects of inferior olive lesion on vestibulo-ocular and optokinetic motor learning.

The combined operation of optokinetic reflex (OKR) and vestibular-ocular reflex (VOR) is essential for image stability during self-motion. Retinal slip signals, which provide neural substrate for OKR and VOR plasticity, are delivered to the inferior olive. Although it has been assumed that the neural circuitry and mechanisms underlying OKR and VOR plasticity are shared, differential role of the inferior olive in the plasticity of OKR and VOR has not been clearly established. To investigate the differential effect of inferior olive lesion on OKR and VOR plasticity, we examined the change of OKR and VOR gains after gain-up and gain-down VOR training. The results demonstrated that inferior olive-lesion differentially affected cerebellum-dependent motor learning. In control mice, OKR gain increased after both gain-up and gain-down VOR training, and VOR gain increased after gain-up VOR training and decreased after gain-down VOR training. In inferior olive-lesioned mice, OKR gain decreased after both gain-up and gain-down VOR training, and while VOR gain did not significantly change after gain-up VOR training, VOR gain decreased after gain-down VOR training. We suggest that multiple mechanisms of plasticity are differentially involved in VOR and OKR adaptation, and gain-up and gain-down VOR learning rely on different plasticity mechanisms.

Inhibitory effect of Jangkanghwan (Korean traditional food) on experimental ulcerative colitis in mice.

Jangkanghwan (JKH) can delay weight loss in mice, promote weight gain during recovery, and reduce colonic shortening and colon weight. In addition, the murine disease activity index was controlled after treatment using JKH. It can reduce the content of pro-inflammatory factors in serum and expression in tissues, such as interleukin (IL)-6, IL-1ß, tumor necrosis factor-α, interferon-γ, cyclooxygenase-2, and nuclear factor kappa-B; in contrast, the content and expression of IL-10 and the inhibitor of nuclear factor kappa-B kinase-α in the serum and tissues were increased. The mRNA expression of the colitis characteristic biomarker monocyte chemoattractant protein-1 and macrophage inflammatory protein-3α were reduced in colon tissues. Using next-generation sequencing technology, the Bacteroidetes phylum in the JKH group decreased, while the Firmicutes phylum increased, and the number of beneficial bacteria-Bifidobacteriaceae, Lactobacillaceae, and Akkermansiaceae-increased. PRACTICAL APPLICATIONS: JKH is a mixture of colonic healthy foods composed of Atractylodes macrocephala koidzumi, radish leaves, Viscum album var. coloratum, dried Zingiber officinale Roscoe, etc. According to UPLC-Q-TOF MS analysis, JKH consists mainly of 17 active substances, such as pheophorbide A, nabumetone alcohol, dehydrocostus lactone, plantamajoside, kaempferol 3, 7-dirhamnoside, quercetin 3-D-glucuronide, and viscumneoside III. We investigated the preventive effects of JKH on dextran sulfate sodium (DSS)-induced ulcerative colitis in a murine model and found that JKH can reduce the damage in mice caused by DSS treatment.

Anti-inflammatory effects of Beopje curly dock (Rumex crispus L.) in LPS-induced RAW 264.7 cells and its active compounds.

Inflammation is a defense response of the body to stimuli. Curly dock (CD) is an herbal food with anti-inflammatory effects. Beopje is an herbal food processing method that reduces toxicity and enhances beneficial effects. This study investigated the effects of CD and Beopje curly dock (CD-B) extracts on lipopolysaccharide (LPS)-induced inflammatory damage in RAW 264.7 cells. Cell survival rate and nitrite concentration were determined using the MTT assay and Griess method, respectively. Enzyme-linked immunosorbent assay was used to detect the inflammatory cytokine levels. The mRNA and protein expression levels of inflammatory associated genes were detected by qPCR and Western blot, respectively. CD and CD-B extracts compositions were assessed by UPLC-Q-TOF MS analysis. Our results indicate that CD-B has a more significant inhibitory effect on the LPS-induced inflammatory response in RAW 264.7 cells than CD, suggesting that the Beopje process potentially enhances the anti-inflammatory effect of CD. PRACTICAL APPLICATIONS: Long-term inflammation can cause a variety of chronic diseases. Therefore, it is necessary to suppress the occurrence of body inflammation in time. This study preliminarily clarified the mechanism of herbal foods to alleviate inflammation by regulating the immune response, and further confirms that applying the Beopje process enhances the anti-inflammatory effect. This research can serve as a significant reference for future research, prevention and treatment of inflammation-related diseases, and the development of functional foods with anti-inflammatory activity. It also provides a theoretical basis for the further reasonable application of Beopje processing method.

Direct translation of climbing fiber burst-mediated sensory coding into post-synaptic Purkinje cell dendritic calcium.

Climbing fibers (CFs) generate complex spikes (CS) and Ca2+ transients in cerebellar Purkinje cells (PCs), serving as instructive signals. The so-called 'all-or-none' character of CSs has been questioned since the CF burst was described. Although recent studies have indicated a sensory-driven enhancement of PC Ca2+ signals, how CF responds to sensory events and contributes to PC dendritic Ca2+ and CS remains unexplored. Here, single or simultaneous Ca2+ imaging of CFs and PCs in awake mice revealed the presynaptic CF Ca2+ amplitude encoded the sensory input's strength and directly influenced post-synaptic PC dendritic Ca2+ amplitude. The sensory-driven variability in CF Ca2+ amplitude depended on the number of spikes in the CF burst. Finally, the spike number of the CF burst determined the PC Ca2+ influx and CS properties. These results reveal the direct translation of sensory information-coding CF inputs into PC Ca2+, suggesting the sophisticated role of CFs as error signals.

Excitatory neuron-specific SHP2-ERK signaling network regulates synaptic plasticity and memory.

Mutations in RAS signaling pathway components cause diverse neurodevelopmental disorders, collectively called RASopathies. Previous studies have suggested that dysregulation in RAS-extracellular signal-regulated kinase (ERK) activation is restricted to distinct cell types in different RASopathies. Some cases of Noonan syndrome (NS) are associated with gain-of-function mutations in the phosphatase SHP2 (encoded by PTPN11); however, SHP2 is abundant in multiple cell types, so it is unclear which cell type(s) contribute to NS phenotypes. Here, we found that expressing the NS-associated mutant SHP2D61G in excitatory, but not inhibitory, hippocampal neurons increased ERK signaling and impaired both long-term potentiation (LTP) and spatial memory in mice, although endogenous SHP2 was expressed in both neuronal types. Transcriptomic analyses revealed that the genes encoding SHP2-interacting proteins that are critical for ERK activation, such as GAB1 and GRB2, were enriched in excitatory neurons. Accordingly, expressing a dominant-negative mutant of GAB1, which reduced its interaction with SHP2D61G, selectively in excitatory neurons, reversed SHP2D61G-mediated deficits. Moreover, ectopic expression of GAB1 and GRB2 together with SHP2D61G in inhibitory neurons resulted in ERK activation. These results demonstrate that RAS-ERK signaling networks are notably different between excitatory and inhibitory neurons, accounting for the cell type-specific pathophysiology of NS and perhaps other RASopathies.

Rumex crispus and Cordyceps militaris Mixture Ameliorates Production of Pro-Inflammatory Cytokines Induced by Lipopolysaccharide in C57BL/6 Mice Splenocytes.

Rumex crispus (Rc) and Cordyceps militaris (Cm) mixture (Rc-Cm; AST2017-01) ameliorated production of proinflammatory cytokines, inflammation-related genes, and nitric oxide (NO) induced by lipopolysaccharide (LPS) in mouse splenocytes. Rc-Cm (6:4) and Taemyeongcheong (commercial healthy drink containing Rc-Cm) were co-administered along with LPS. Rc-Cm inhibited production of tumor necrosis factor α, interferon γ, interleukin (IL)-1ß, and IL-6 in LPS-induced splenocytes. However, levels of inflammatory cytokines were elevated in the absence of LPS treatment. Rc-Cm significantly suppressed mRNA expression of IL-1ß, IL-6, and the inflammation-related genes inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2), as well as NO production upon LPS co-treatment. Whereas Rc-Cm increased mRNA expression of IL-1ß, and IL-6, but did not up-regulate expression of iNOS and COX-2, or increase NO production without LPS co-treatment. Therefore, treatment of Rc-Cm to LPS-induced splenocytes ameliorated induction of pro-inflammatory cytokines, inflammation-related genes, and NO production. In the absence of LPS, Rc-Cm treatment up-regulated pro-inflammatory cytokines but did not alter expression of the inflammation-related genes iNOS and COX-2 or NO production. These results indicate that the natural phytochemicals chrysophanol and cordycepin in Rc-Cm promote anti-inflammatory activities and immune cell responses.