Naringin alleviates pneumonia caused by Klebsiella pneumoniae infection by suppressing NLRP3 inflammasome.

Klebsiella pneumoniae (Kpn) is an important pathogen of hospital-acquired pneumonia, which can lead to sepsis and death in severe cases. In this study, we simulated pneumonia induced by Kpn infection in mice to investigate the therapeutic effect of naringin (NAR) on bacterial-induced lung inflammation. Mice infected with Kpn exhibited increases in white blood cells (WBC) and neutrophils in the peripheral blood and pathological severe injury of the lungs. This injury was manifested by increased expression of the inflammatory cytokines interleukin (IL)- 18, IL-1ß, tumor necrosis factor-α (TNF-α) and IL-6, and elevated the expression of NLRP3 protein. NAR treatment could decrease the protein expression of NLRP3, alleviate lung inflammation, and reduce lung injury in mice caused by Kpn. Meanwhile, molecular docking results suggest NAR could bind to NLRP3 and Surface Plasmon Resonance (SPR) analyses also confirm this result. In vitro trials, we found that pretreated with NAR not only inhibited nuclear translocation of nuclear factor (NF)-κB protein P65 but also attenuated the protein interaction of NLRP3, caspase-1 and ASC and inhibited the assembly of NLRP3 inflammasome in mice AMs. Additionally, NAR could reduce intracellular potassium (K+) efflux, inhibiting NLRP3 inflammasome activation. These results indicated that NAR could protect against Kpn-induced pneumonia by inhibiting the overactivation of the NLRP3 inflammasome signaling pathway. The results of this study confirm the efficacy of NAR in treating bacterial pneumonia, refine the mechanism of action of NAR, and provide a theoretical basis for the research and development of NAR as an anti-inflammatory adjuvant.

Screening of essential oils with acaricidal activity against Haemaphysalis longicornis (Acari: Ixodidae) and analysis of active components.

Haemaphysalis longicornis (Acari: Ixodidae) is an important vector of numerous pathogens and poses a great threat to veterinary and public health. Commercially available tick repellents are extensively used and primarily comprise synthetic molecules; however, there are concerns over their safety and environmental impacts. Biologically based acaricides, particularly the plant-derived essential oils (EOs), may constitute an appealing alternative. We screened 20 different EOs by packet tests of unfed H. longicornis nymphs, and found that EOs of cinnamon, clove and chamomile were the most toxic (mortality > 80 %). Cinnamon EO had the most competitive acaricidal activity, with lethal concentration 50 (LC50) rates of 0.4530 %, 0.2316 % and 0.0342 % (v/v) for unfed adults, nymphs and larvae, respectively. Furthermore, 5.00 % (v/v) cinnamon EO showed reproductive inhibition against H. longicornis, with significantly higher rates of oviposition reduction (53.19 %) and hatching reduction (46.21 %) compared with the negative control group. Composition analysis of cinnamon EO by gas chromatography-mass spectrometry (GC-MS) revealed that the major chemical compounds were trans-cinnamaldehyde (72.21 %) and cinnamic acid (19.45 %), with the former showing similar levels of acaricidal activity and oviposition inhibition as cinnamon EO. This study has demonstrated the potential of cinnamon EO and trans-cinnamaldehyde as natural acaricides against H. longicornis, and is the first to characterize their oviposition inhibition activity.

Phillygenin activates PKR/eIF2α pathway and induces stress granule to exert anti-avian infectious bronchitis virus.

The prevalence of avian infectious bronchitis virus (IBV) is still one of causes inducing severe losses of production in the poultry industry worldwide. Vaccination does not completely prevent IBV infection and spread due to immune failure and viral mutations. ForsythiaeFructus and its compounds have been widely used in a lot of prescriptions of the traditional Chinese medicine for a long history, and it is well-known as safety and efficiency in heat-clearing and detoxifying. This study aims to investigate the anti-IBV activity and mechanism of phillygenin. The results showed that phillygenin inhibited IBV replication by disturbing multiple stages of the virus life cycle, including viral adsorption, invasion, internalization, and release in Vero cells. After being treated with 100, 125 and 150 µg/mL phillygenin, the expression of G3BP1 was significantly increased and the phosphorylation of PKR/eIF2α was activated, which increased stress granule, thereby triggering the antiviral response in Vero cells. The anti-virus activity of PHI was decreased when G3BP1 was interfered by si-RNA, and G3BP1 was down-regulated when PKR/eIF2α was interfered by si-RNA. In conclusion, our findings indicate that phillygenin activates PKR/eIF2α pathway and induces stress granule formation to exert anti-IBV, which holds promise to develop into a novel anti-IBV drug. Further study in vivo is needed to explore phillygenin as a potential and effective drug to prevent IB in poultry.

Huang Bai Jian Pi decoction alleviates diarrhea and represses inflammatory injury via PI3K/Akt/NF-κB pathway: In vivo and in vitro studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Huang Bai Jian Pi (HBJP) decoction, a Chinese herbal formula based on the Pulsatilla decoction (PD) and Si Junzi decoction, is efficacy to treat clinical diarrhea in calves. AIM OF THE STUDY: The mechanism of HBJP decoction to treat calf diarrhea remains unclear. This study was to investigate the therapeutic effect and anti-inflammatory mechanism of HBJP decoction on diarrhea in rats. MATERIALS AND METHODS: Thirty-six Sprague Dawley rats were randomly divided into control group, model group, PD group and three treated groups with HBJP decoction. The diarrheal model in rats was established by multiple factors including high-sugar and fat diet, high temperature and dampness environment, biological pathogenic factors. The diarrheal animals were treated with HBJP decoction or PD for 5 days. The inflammatory model of the intestinal epithelioid cell line 6 (IEC-6) was induced by TNF-α. The clinical symptoms, blood routine and biochemistry parameters, histopathology of main organs were detected. The proteins associated with PI3K/Akt/NF-κB pathway and the expression levels of cytokines associated with inflammation were detected in vivo and in vitro by Western blot and ELISA. RESULTS: The model rats showed obvious diarrheal symptoms, and the obvious systemic inflammatory response accompanied with abnormal change in blood routine, biochemistry parameters and histopathology. HBJP decoction alleviated obviously the clinical symptoms, and pathological changes of the liver, colon and lung, and abnormal blood routine and biochemistry indexes in rats. The expression of P-PI3K, P-Akt, P-NF-κB, IL-1ß, IL-6 was significantly increased, and the expression of IL-10 was markedly decreased in diarrheal rats and IEC-6 with inflammation. HBJP decoction significantly inhibited the PI3K/AKT/NF-κB signal pathway and adjusted the expression of these inflammatory cytokines. CONCLUSIONS: The finding suggested that HBJP decoction alleviate the inflammation in diarrhea through inhibiting the PI3K/Akt/NF-κB signal pathway, which provides scientific evidences for the clinical application of HBJP decoction in diarrhea.

The Expressing Patterns of Opioid Peptides, Anti-opioid Peptides and Their Receptors in the Central Nervous System Are Involved in Electroacupuncture Tolerance in Goats.

To investigate dynamic processes of enkephalin (ENK), cholecystokinin octapeptide (CCK-8), orphanin FQ (OFQ) and their receptors (µ opioid receptor, MOR; CCK B type receptor, CCKBR and opioid receptor-like 1 receptor, OPRL1) in the central nerve system (CNS) during electroacupuncture (EA) tolerance, EA of Sixty Hz was used to stimulate goats for 6 h. Pain threshold was measured using potassium iontophoresis. The expression levels of ENK, CCK-8, and OFQ and their receptors were determined with ELISA and qPCR, respectively. The results showed that the change rates of pain threshold in EA-treated goats decreased from 89.9 ± 11.7% at 0.5 h to -11.4 ± 8.9% at 6 h. EA induced the decreased ENK and increased CCK-8 and OFQ in the most measured nuclei. EA caused decreased preproenkephalin mRNAs in ACB, CAU, PVH, and PAG at 4 h, and decreased or unchanged MOR mRNAs at 2-6 h, but increased CCK mRNAs in CAU, PVT, PVH, PAG, and SCD at 4-12 h. Increased prepronociceptin mRNAs and fluctuated CCKBR and OPLR1 mRNAs were found in the most measured nuclei. ENK levels were positively correlated (p < 0.01) with the change rates of pain thresholds in the measured nuclei or areas while CCK-8 levels (or OFQ levels) were negatively correlated (p < 0.01) with the pain thresholds in CAU (or CAU and ACB). These results suggest that the development and recovery of EA tolerance may be associated with the specific expression patterns of opioid peptides, anti-opioid peptides and their receptors in the analgesia-related nuclei or areas.

MiRNAs are involved in chronic electroacupuncture tolerance in the rat hypothalamus.

Acupuncture tolerance is the gradual decrease in analgesic effect due to its prolonged application. However, its mechanism in terms of miRNA is still unknown. To explore the role of miRNAs in electroacupuncture (EA) tolerance of rats using deep sequencing, rats with more than a 50 % increase in tail flick latency (TFL) in response to EA were selected for this experiment. EA tolerance was induced by EA once daily for eight consecutive days. The hypothalami were harvested for deep sequencing. As a result, 49 differentially expressed miRNAs were identified and validated by real-time PCR. Of them, let-7b-5p, miR-148a-3p, miR-124-3p, miR-107-3p, and miR-370-3p were further confirmed to be related to EA tolerance by an intracerebroventricular injection of agomirs or antagomirs of these miRNAs. Potential targets of the 49 miRNAs were enriched in 9 pathways and 282 gene ontology (GO) terms. Five miRNAs were confirmed to participate in EA tolerance probably through the functional categories related to nerve impulse transmission, receptor signal pathways, and gene expression regulation, as well as pathways related to MAPK, neurotrophin, fatty acid metabolism, lysosome, and the degradation of valine, leucine, and isoleucine. Our findings reveal a characterized panel of the differentially expressed miRNAs in the hypothalamus in response to EA and thus provide a solid experimental framework for future analysis of the mechanisms underlying EA-induced tolerance.

Analgesic Neural Circuits Are Activated by Electroacupuncture at Two Sets of Acupoints.

To investigate analgesic neural circuits activated by electroacupuncture (EA) at different sets of acupoints in the brain, goats were stimulated by EA at set of Baihui-Santai acupoints or set of Housanli acupoints for 30 min. The pain threshold was measured using the potassium iontophoresis method. The levels of c-Fos were determined with Streptavidin-Biotin Complex immunohistochemistry. The results showed pain threshold induced by EA at set of Baihui-Santai acupoints was 44.74% ± 4.56% higher than that by EA at set of Housanli acupoints (32.64% ± 5.04%). Compared with blank control, EA at two sets of acupoints increased c-Fos expression in the medial septal nucleus (MSN), the arcuate nucleus (ARC), the nucleus amygdala basalis (AB), the lateral habenula nucleus (HL), the ventrolateral periaqueductal grey (vlPAG), the locus coeruleus (LC), the nucleus raphe magnus (NRM), the pituitary gland, and spinal cord dorsal horn (SDH). Compared with EA at set of Housanli points, EA at set of Baihui-Santai points induced increased c-Fos expression in AB but decrease in MSN, the paraventricular nucleus of the hypothalamus, HL, and SDH. It suggests that ARC-PAG-NRM/LC-SDH and the hypothalamus-pituitary may be the common activated neural pathways taking part in EA-induced analgesia at the two sets of acupoints.

The Expression Patterns of c-Fos and c-Jun Induced by Different Frequencies of Electroacupuncture in the Brain.

To investigate patterns of c-Fos and c-Jun expression induced by different frequencies of electroacupuncture (EA) in the brain, goats were stimulated by EA of 0, 2, 60, or 100 Hz at a set of "Baihui, Santai, Ergen, and Sanyangluo" points for 30 min. The pain threshold was measured using the potassium iontophoresis method. The levels of c-Fos and c-Jun were determined with Streptavidin-Biotin Complex immunohistochemistry. The results showed that the pain threshold induced by 60 Hz was 82.2% higher (P < 0.01) than that by 0, 2, or 100 Hz (6.5%, 35.2%, or 40.9%). EA induced increased c-Fos and c-Jun expression in most analgesia-related nuclei and areas in the brain. Sixty Hz EA increased more c-Fos or c-Jun expression than 2 Hz or 100 Hz EA in all the measured nuclei and areas except for the nucleus accumbens, the area septalis lateralis, the caudate nucleus, the nucleus amygdala basalis, and the locus coeruleus, in which c-Fos or c-Jun expressions induced by 60 Hz EA did not differ from those by 2 Hz or 100 Hz EA. It was suggested that 60 Hz EA activated more extensive neural circuits in goats, which may contribute to optimal analgesic effects.

Electroacupuncture-induced dynamic processes of gene expression levels of endogenous opioid Peptide precursors and opioid receptors in the CNS of goats.

In order to investigate the dynamic processes of mRNA levels of proenkephalin, proopiomelanocortin, prodynorphin, and opioid receptors (δ-, µ-, and κ-receptor) induced by electroacupuncture (EA) in the central nerve system, goats were stimulated by EA of 60 Hz for 0.5 h at a set of Baihui, Santai, Ergen, and Sanyangluo points. The pain threshold was measured using the method of potassium iontophoresis. The mRNA levels of the three opioid peptide precursors and three opioid receptors were determined with quantitative real-time PCR and the levels of Met-enkephalin with SABC immunohistochemistry at 0.5 h before and at 0, 2, 4, 6, 8, 12, and 24 h after EA. The results showed that the pain threshold correlated (P < 0.01) with Met-enkephalin immunoactivities in the measured nuclei and areas of goats. The analgesic aftereffect lasted for 12 h at least. The mRNA levels of the three opioid peptide precursors and three opioid receptors began to increase at 0 h, reached the peak during the time from 4 h to 6 h or at 12 h, and remained higher at 24 h after EA was discontinued. These results suggested that the initiation of gene expression of opioid peptides and the three receptors may be associated with EA-induced analgesic aftereffect.

Effects of electroacupuncture of different frequencies on the release profile of endogenous opioid peptides in the central nerve system of goats.

To investigate the release profile of met-enkephalin, ß-endorphin, and dynorphin-A in ruminants' CNS, goats were stimulated by electroacupuncture of 0, 2, 40, 60, 80, or 100 Hz for 30 min. The pain threshold was measured using potassium iontophoresis. The peptide levels were determined with SABC immunohistochemisty. The results showed that 60 Hz increased pain threshold by 91%; its increasing rate was higher (P < 0.01) than any other frequency did. 2 Hz and 100 Hz increased met-enkephalin immunoactivities (P < 0.05) in nucleus accumbens, septal area, caudate nucleus, amygdala, paraventricular nucleus of hypothalamus, periaqueductal gray, dorsal raphe nucleus, and locus ceruleus. The two frequencies elicited ß-endorphin release (P < 0.05) in nucleus accumbens, septal area, supraoptic nucleus, ventromedial nucleus of hypothalamus, periaqueductal gray, dorsal raphe nucleus, locus ceruleus, solitary nucleus and amygdala. 60 Hz increased (P < 0.05) met-enkephalin or ß-endorphin immunoactivities in the nuclei and areas mentioned above, and habenular nucleus, substantia nigra, parabrachial nucleus, and nucleus raphe magnus. High frequencies increased dynorphin-A release (P < 0.05) in spinal cord dorsal horn and most analgesia-related nuclei. It suggested that 60 Hz induced the simultaneous release of the three peptides in extensive analgesia-related nuclei and areas of the CNS, which may be contributive to optimal analgesic effects and species variation.