Niuhuang Qingxin Wan ameliorates depressive-like behaviors and improves hippocampal neurogenesis through modulating TrkB/ERK/CREB signaling pathway in chronic restraint stress or corticosterone challenge mice.

Introduction: Chronic stress-associated hormonal imbalance impairs hippocampal neurogenesis, contributing to depressive and anxiety behaviors. Targeting neurogenesis is thus a promising antidepressant therapeutic strategy. Niuhuang Qingxin Wan (NHQXW) is an herbal formula for mental disorders in Traditional Chinese Medicine (TCM) practice, but its anti-depressant efficacies and mechanisms remain unverified. Methods: In the present study, we tested the hypothesis that NHQXW could ameliorate depressive-like behaviors and improve hippocampal neurogenesis by modulating the TrkB/ERK/CREB signaling pathway by utilizing two depression mouse models including a chronic restraint stress (CRS) mouse model and a chronic corticosterone (CORT) stress (CCS) induced mouse model. The depression-like mouse models were orally treated with NHQXW whereas fluoxetine was used as the positive control group. We evaluated the effects of NHQXW on depressive- and anxiety-like behaviors and determined the effects of NHQXW on inducing hippocampal neurogenesis. Results: NHQXW treatment significantly ameliorated depressive-like behaviors in those chronic stress mouse models. NHQXW significantly improved hippocampal neurogenesis in the CRS mice and CCS mice. The potential neurogenic mechanism of NHQXW was identified by regulating the expression levels of BDNF, TrkB, p-ERK (T202/T204), p-MEK1/2 (S217/221), and p-CREB (S133) in the hippocampus area of the CCS mice. NHQXW revealed its antidepressant and neurogenic effects that were similar to fluoxetine. Moreover, NHQXW treatment revealed long-term effects on preventing withdrawal-associated rebound symptoms in the CCS mice. Furthermore, in a bioactivity-guided quality control study, liquiritin was identified as one of the bioactive compounds of NHQXW with the bioactivities of neurogenesis-promoting effects. Discussion: Taken together, NHQXW could be a promising TCM formula to attenuate depressive- and anxiety-like behaviors against chronic stress and depression. The underlying anti-depressant mechanisms could be correlated with its neurogenic activities by stimulating the TrkB/ERK/CREB signaling pathway.

Acteoside promotes B cell-derived IL-10 production and ameliorates autoimmunity.

IL-10-producing regulatory B (Breg) cells are well recognized for maintaining immune tolerance. The impaired Breg cell function with decreased IL-10-producing capacity has been found in autoimmune diseases, such as rheumatoid arthritis, lupus, and primary Sjogren's syndrome (pSS). However, seldom therapeutic agents targeting Breg cells are available to treat those autoimmune diseases. Here, we showed that acteoside (AC), a caffeoyl phenylethanoid glycoside from a medicinal herb Radix Rehmanniae, could promote IL-10 production from both human and murine B cells via critically regulating the TLR4/PI3K axis. Moreover, TLR4 was found increased in Breg cells from mice with experimental SS (ESS), a mouse model that recapitulates human pSS. Thus, B cells from the ESS mice were susceptible to AC treatment, showing higher IL-10-producing capacity than those from naïve controls. In addition, AC treatment also promoted the production of IL-10 from TLR4+ CXCR4+ plasma cells of ESS mice. Notably, we found that AC was able to enter lymphoid organs upon oral administration. AC treatment effectively increased IL-10+ B cells in ESS mice and ameliorated disease pathology accompanied by reduced T effector cells, including Th17 and T follicular helper cells in the ESS mice. In conclusion, AC could promote Breg cell function and attenuate ESS pathology in vivo, which may be a promising drug candidate for treating pSS and other autoimmune diseases.

Rehmapicroside ameliorates cerebral ischemia-reperfusion injury via attenuating peroxynitrite-mediated mitophagy activation.

Peroxynitrite (ONOO-)-mediated mitophagy activation represents a vital pathogenic mechanism in ischemic stroke. Our previous study suggests that ONOO- mediates Drp1 recruitment to the damaged mitochondria for excessive mitophagy, aggravating cerebral ischemia/reperfusion injury and the ONOO--mediated mitophagy activation could be a crucial therapeutic target for improving outcome of ischemic stroke. In the present study, we tested the neuroprotective effects of rehmapicroside, a natural compound from a medicinal plant, on inhibiting ONOO--mediated mitophagy activation, attenuating infarct size and improving neurological functions by using the in vitro cultured PC12 cells exposed to oxygen glucose deprivation with reoxygenation (OGD/RO) condition and the in vivo rat model of middle cerebral artery occlusion (MCAO) for 2 h of transient cerebral ischemia plus 22 h of reperfusion. The major discoveries include following aspects: (1) Rehmapicroside reacted with ONOO- directly to scavenge ONOO-; (2) Rehmapicroside decreased O2- and ONOO-, up-regulated Bcl-2 but down-regulated Bax, Caspase-3 and cleaved Caspase-3, and down-regulated PINK1, Parkin, p62 and the ratio of LC3-II to LC3-I in the OGD/RO-treated PC12 cells; (3) Rehmapicroside suppressed 3-nitrotyrosine formation, Drp1 nitration as well as NADPH oxidases and iNOS expression in the ischemia-reperfused rat brains; (4) Rehmapicroside prevented the translocations of PINK1, Parkin and Drp1 into the mitochondria for mitophagy activation in the ischemia-reperfused rat brains; (5) Rehmapicroside ameliorated infarct sizes and improved neurological deficit scores in the rats with transient MCAO cerebral ischemia. Taken together, rehmapicroside could be a potential drug candidate against cerebral ischemia-reperfusion injury, and its neuroprotective mechanisms could be attributed to inhibiting the ONOO--mediated mitophagy activation.

Calycosin Influences the Metabolism of Five Probe Drugs in Rats.

BACKGROUND: Calycosin (CAL), a type of O-methylated isoflavone extracted from the herb Astralagusmembranaceus (AM), is a bioactive chemical with antioxidative, antiphlogistic and antineoplastic activities commonly used in traditional alternative Chinese medicine. AM has been shown to confer health benefits as an adjuvant in the treatment of a variety of diseases. AIM: The main objective of this study was to determine whether CAL influences the cytochrome P450 (CYP450) system involved in drug metabolism. METHODS: Midazolam, tolbutamide, omeprazole, metoprolol and phenacetin were selected as probe drugs. Rats were randomly divided into three groups, specifically, 5% Carboxymethyl cellulose (CMC) for 8 days (Control), 5% CMC for 7 days + CAL for 1 day (single CAL) and CAL for 8 days (conc CAL), and metabolism of the five probe drugs evaluated using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). RESULTS: No significant differences were observed for omeprazole and midazolam, compared to the control group. T max and t1/2 values of only one probe drug, phenacetin, in the conc CAL group were significantly different from those of the control group (T max h: 0.50±0.00 vs 0.23±0.15; control vs conc CAL). C max of tolbutamide was decreased about two-fold in the conc CAL treatment group (conc vs control: 219.48 vs 429.56, P<0.001). CONCLUSION: Calycosin inhibits the catalytic activities of CYP1A2, CYP2D6 and CYP2C9. Accordingly, we recommend caution, particularly when combining CAL as a modality therapy with drugs metabolized by CYP1A2, CYP2D6 and CYP2C9, to reduce the potential risks of drug accumulation or ineffective treatment.

Tetrandrine Ameliorates Airway Remodeling of Chronic Asthma by Interfering TGF-ß1/Nrf-2/HO-1 Signaling Pathway-Mediated Oxidative Stress.

Background: Imbalanced oxidative stress and antioxidant defense are involved in airway remodeling in asthma. It has been demonstrated that Tetrandrine has a potent role in antioxidant defense in rheumatoid arthritis and hypertension. However, the correlation between Tetrandrine and oxidative stress in asthma is utterly blurry. This study aimed to investigate the role of Tetrandrine on oxidative stress-mediated airway remolding. Materials and Methods: Chronic asthma was established by ovalbumin (OVA) administration in male Wistar rats. Histopathology was determined by HE staining. Immunofluorescence was employed to detect the expression of α-SMA and Nrf-2. Level of oxidative stress and matrix metalloproteinases were examined by ELISA kits. Cell viability and cell cycle of primary airway smooth muscle cells (ASMCs) were evaluated by CCK8 and flow cytometry, respectively. Signal molecules were detected using western blot. Results: Tetrandrine effectively impairs OVA-induced airway inflammatory and airway remodeling by inhibiting the expression of CysLT1 and CysLTR1. The increase of oxidative stress and subsequent enhancement of MMP9 and TGF-ß1 expression were rescued by the administration of Tetrandrine in the rat model of asthma. In in vitro experiments, Tetrandrine markedly suppressed TGF-ß1-evoked cell viability and cell cycle promotion of ASMCs in a dose-dependent manner. Furthermore, Tetrandrine promoted Nrf-2 nuclear transcription and activated its downstream HO-1 in vivo and in vitro. Conclusion: Tetrandrine attenuates airway inflammatory and airway remodeling in rat model of asthma and TGF-ß1-induced cell proliferation of ASMCs by regulating oxidative stress in primary ASMCs, suggesting that Tetrandrine possibly is an effective candidate therapy for asthma.

Evaluation of lentinan effects on cytochrome P450 activity in rats by a cocktail method.

OBJECTIVES: In this study, a cocktail of probe drugs was used to assess whether lentinan could influence the activities of rat enzymes CYP3A4, CYP2D6, CYP1A2, CYP2C19, and CYP2C9 in vivo. MATERIALS AND METHODS: Fourteen days after intraperitoneal injection of lentinan, rats were given an oral dose of a cocktail solution containing phenacetin, tolbutamide, omeprazole, metoprolol, and midazolam. Then, we obtained blood in specific durations for the determination of plasma concentration of the probe drugs using UPLC-MS/MS. We also evaluated the pharmacokinetic parameters using the DAS 2.0 software. RESULTS: We found that various concentrations of lentinan increased the activity of rat CYP1A2, CYP3A4, CYP2D6, and CYP2C19 but not CYP2C9. CONCLUSION: These findings suggest that clinical application of lentinan combination with CYP3A4, CYP1A2, CYP2C19, or CYP2D6 should be given careful consideration as this may lead to herb-drug interactions and hence treatment failure.

Short-term high salt intake impairs hepatic mitochondrial bioenergetics and biosynthesis in SIRT3 knockout mice.

High salt intake (HS) is an important factor in the development of many metabolic diseases. The liver is the metabolic center in the body. However, the effect of short-term HS on the liver mitochondria and its mechanism are still unclear. In this study, we investigated the effects of short-term HS on liver mitochondrial function. We found that HS reduced Sirtuin3 (SIRT3) protein level, increasing protein carbonylation in mice liver. HS intake decreased ATP production, mitochondrial transcription factor A (TFAM), and complex I level. SIRT3 knockout (SKO) mice exhibited similar results with HS-treated wild-type mice but with a less extent of carbonylation and ATP reduction. Our study shows that short-term HS led to increased hepatic oxidative state, impaired mitochondrial biosynthesis, and bioenergetics. HS-treated mice could still maintain hepatic glucose homeostasis by compensatory activation of Adenosine 5'-monophosphate-activated protein kinase (AMPK). However, in HS-treated SKO mice, AMPK was not activated, instead, the glycogen synthase activity increased, which caused an exceptionally increased glycogen accumulation. This study provides evidence that short-term HS intake could cause the early hepatic metabolic changes, highlighting the importance of controlling salt intake especially in those patients with defects in SIRT3. Highlights High salt intake down-regulates SIRT3 protein level and increases oxidation. High salt intake activates AMPK via AMP-dependent pathway. High salt intake impairs energy metabolism. High salt combined with SIRT3 knockout results in glycogen accumulation.

Effects of salidroside on rat CYP enzymes by a cocktail of probe drugs.

OBJECTIVES: In this study, we aimed to evaluate the effect of salidroside on the activities of the different drug-metabolizing enzymes CYP1A2, CYP2B6, CYP2C9, CYP2D6 and CYP3A4 in rats, in which a specific probe drug was used for each enzyme. MATERIALS AND METHODS: After pretreatment with salidroside, five probe drugs were simultaneously administered to rats by gavage. The given dose was 2.0 mg/kg for phenacetin (CYP1A2 activity), 4.0 mg/kg for bupropion (CYP2B6 activity), 2.0 mg/kg for losartan (CYP2C9 activity), 8.0 mg/kg for metoprolol (CYP2D6 activity) and 1.0 mg/kg for midazolam (CYP3A4 activity). Then, an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to analyze the concentrations of rats' blood, which were collected at different corresponding times. RESULTS: Our data showed that salidroside exhibited an inductive effect on CYP1A2, CYP2B6, CYP2C9 and CYP3A4 activities by changing the main pharmacokinetic parameters (t1/2, CL/F, Cmax and AUC(0-∞)) of the four probe drugs in rats. However, no significant changes in CYP2D6 activity were observed. CONCLUSION: In a word, the results displayed that salidroside could induce the activities of CYP1A2, CYP2B6, CYP2C9 and CYP3A4, which may influence the disposition of the drugs that are mainly metabolized by these pathways. Our research can provide the basis for the study of related herb-drug interactions in clinic.

Neuroanatomical characteristics of deep and superficial needling using LI11 as an example.

OBJECTIVES: To compare the neuroanatomical characteristics of the deep and superficial tissues at acupuncture point LI11 using a neural tracing technique, in order to examine the neural basis of potential differences between deep and superficial needling techniques. METHODS: In order to mimic the situations of the deep and superficial needling, the retrograde neural tracer Alexa Fluor 488 conjugate of cholera toxin subunit B (AF488-CTB) was injected into the muscle or subcutaneous tissue, respectively, at acupuncture point LI11 in eight rats (n=4 each). Three days following injection, the distribution of motor and sensory neurons labelled with AF488-CTB was examined in the spinal cord and dorsal root ganglia (DRG) under a fluorescent microscope. RESULTS: For both types of injection, labelled motor and sensory neurons were distributed on the side ipsilateral to the injection in the spinal cord and DRG between spinal levels C5 and T1. The number of labelled motor neurons following intramuscular injection was significantly higher than subcutaneous injection. By contrast, the number of labelled sensory neurons following subcutaneous injection was significantly higher in number and extended over a greater number of spinal segments compared to intramuscular injection. CONCLUSIONS: These data indicate that the motor and sensory innervation of muscle and subcutaneous tissue beneath LI11 differ, and suggest that acupuncture signals induced by deep and superficial needling stimulation may be transmitted through different neural pathways.

Local cutaneous nerve terminal and mast cell responses to manual acupuncture in acupoint LI4 area of the rats.

Previous studies have shown that the effects of manual acupuncture (MA) are contributed by collagen fibers and mast cells in local acupoints, at which acupuncture stimulation causes various afferent fiber groups to be excited. However what happens in local nerve fibers and mast cells after MA remains unclear. The aim of this study was to examine the response of cutaneous nerve fibers and mast cells to MA stimulation in acupoint Hegu (LI4). The contralateral LI4 of the same rat was used as a non-stimulated control. Immnohistochemistry analysis were carried out to observe the expression of histamine (HA), serotonin (5-HT) and nociceptive neuropeptides, calcitonin gene-related peptide (CGRP) and substance P (SP), in the LI4 area. Mast cells were labeled with anti-mast cell tryptase antibody and simultaneously with HA or 5-HT primary antibodies to observe their co-expression. Our results showed that SP and CGRP were expressed more highly on the cutaneous nerve fibers of LI4 after MA stimulation than that of the control. Mast cells aggregated in close proximity to the blood vessels in intra-epidermis and dermis and some of them with degranulation in the lower dermis and subcutaneous tissue of LI4. Both mast cells and their granules appeared with HA (+) and 5-HT (+) expression at stimulated L14 sites, while a few intact mast cells with a little expression of 5-HT and HA were distributed in areas of non-stimulated L14. The results indicated that local cutaneous nerve terminals and mast cells responded to MA with higher expression of SP and CGRP in nerve fibers, as well as with aggregation and degranulation of mast cells with HA and 5-HT granules at acupoint LI4. These neuroactive substances may convey signals to certain pathways that contribute to the effects of acupuncture.

[Entity of acupoint: kinetic changes of acupoints in histocytochemistry].

The present study aims to investigate the kinetic histocytochemical changes of acupoints in different condition. The expression of tryptase (+) mast cells, histamine (HA) , serotonin (5-HT) and nociceptive neuropeptides including calcitonin gene-related peptide (CGRP) and substance P (SP) were observed by immunohistochemistry combined with confocal technology. Mast cells were labeled with anti-mast cell tryptase antibody and simultaneously with HA or 5-HT primary antibodies to observe their co-expression. The results showed that: (1) SP and CGRP were expressed more highly on the cutaneous nerve fibers of "Hegu" (LI 4) after acupuncture stimulation than that of the control. Mast cells aggregated in close proximity to the blood vessels in intra-epidermis and dermis, and some of them with degranulation in the lower dermis and subcutaneous tissue of "Hegu" (LI 4). Both mast cells and their granules appeared with HA (+) and 5-HT (+) expression at stimulated LI 4 sites, while a few intact mast cells with a little expression of 5-HT and HA were distributed in areas of non-stimulated Ll 4. (2) The acupoints in different locations such as Baihui (GV 20), Weishu (BL 21), Zhongwan (CV 12) and LI 4 had the same constituent but the contents were different. (3) The histocytochemical responses of acupoints sensitized by the Gastric mucosa injury (GMI) were also investigated. GMI resulted in neurogenic plasma extravasation by Evans Blue (EB) in the skin of the acupoints over the back and abdomen, which mostly occurred in the T9-T11 dermatomere. The EB extravasation dots just like acupoints sensitization appeared after GMI and disappeared gradually during the natural self-recovery of the gastric mucosa. More SP and CGRP positive nerve fibers were distributed in EB dots than in regions beside EB dots and in the control, mostly distributed in the nerve fibers around both the vessels and root of hair follicle. Mast cells also aggregated and degranulated to release algogenic substances of 5-HT and HA around the vessels in areas of the EB dots. Collectively the acupoints displayed the same histocytochemical responses due to either acupuncture stimulation or GMI. This may potentially be the histocytochemical basis in the local acupoints and acupoints displayed kinetic changes in different condition.

[Distribution of calcitonin gene related peptide positive neurofibers in local skin tissues of "Neiting"(ST 44), "Zusanli" (ST 36) and "Futu" (ST 32) regions in the rat]. / é™é’™ç´ åŸºå› ç›¸å ³è‚½é˜³æ€§è¡¨è¾¾ç¥žç»çº¤ç»´åœ¨å¤§é¼ "å† åº­""足三里""ä¼å ”"穴区皮肤组织中的分布

OBJECTIVE: To observe the distribution of calcitonin gene related peptide (CGRP)-positive neurofibers in local tissues of "Neiting"(ST 44), "Zusanli" (ST 36) and "Futu" (ST 32) regions in the hind limbs of rats. METHODS: Five adult male SD rats were used in the present study. The local skin tissues of acupoint "Neiting" (ST 44), "Zusanli" (ST 36) and "Futu" (ST 32) regions were dissected from the rat that underwent transcardiac perfusion with 4% paraformaldehyde, and cut into sections (20 microm in thickness), followed by staining with CGRP fluorescent immunohistochemical method and with phalloidin and DAPI, respectively. After that, CGRP-positive neurofibers of the samples were observed and recorded using a laser confocal microscope. RESULTS: CGRP-positive nerve fibers were found to distribute in the dermis and subcutaneous layers of local tissues of acupoint ST 44, ST 36 and ST 32, mainly concentrating around the vessel-like structure. The number and length of CGRP-positive nerve fibers were most in the ST 44 region, more in the ST 36 region and fewer in the ST 32 region, in turn (P<0.01). The distribution of the vessel-like structure also presented a decreasing trend in the above-mentioned three acupoint regions in turn. CONCLUSION: CGRP-positive neurofibers are an important element in the local tissues of acupoint ST 44, ST 36 and ST 32 regions, and present a decreasing tendency in the number from the distal to the proximal part of the hind-limb, showing a similar tendency of needling sensation in the human body in which a more hypersensitivity exists in the distal part of the limb.

Manual acupuncture and laser acupuncture for autonomic regulations in rats: observation on heart rate variability and gastric motility.

This study focused on the effects of laser acupuncture (LA) and manual acupuncture (MA) at different acupoints on gastric motility and heart rate variability (HRV) simultaneously to elucidate the site specific effects of acupoints and the correlation between changes of gastric motility and low frequency/high frequency (LF/HF) ratio. Gastric motility and HRV were recorded before and during MA or LA. Stimulating PC-6 or ST-36 significantly enhanced gastric motility, while BL-21 caused no changes. In contrast, MA or LA at CV-12 significantly suppressed gastric motility. Stimulating PC-6 or ST-36 significantly increased heart rate (HR), while CV-12 or BL-21 induced no significant changes of HR. Stimulating PC-6 significantly increased LF/HF, while ST-36, CV-12, or BL-21 induced no significant effects. These results indicated that there was acupoint specificity in the effects of acupuncture on gastric motility and HRV. The stimulatory effect of MA and LA at PC-6 and ST-36 on HR was associated with sympathetic activity. The stimulatory effect of MA or LA at PC-6 or ST-36 on gastric motility was associated with vagal activity. Laser needle can be used as an alternative stimulation therapy.