Bupleurum chinense exerts a mild antipyretic effect on LPS-induced pyrexia rats involving inhibition of peripheral TNF-α production.

ETHNOPHARMACOLOGICAL RELEVANCE: Bupleuri Radix, the dried roots of Bupleurum chinense DC. (BC) or Bupleurum scorzonerifolium Willd., is one of the most frequently used traditional Chinese medicines. As the species in Xiao-Chai-Hu decoction, BC has been used as an antipyretic medicine with a long history. However, its antipyretic characteristics and underlying mechanism(s) remain unclear. AIM OF THE STUDY: To elucidate the antipyretic characteristics and mechanism(s) of BC used in its traditional way. METHODS: The water extract of BC (BCE) was prepared according to the traditional decocting mode. Murine fever and endotoxemia models were induced by intravenous injection of lipopolysaccharide (LPS). In vitro complement activation assay and the levels of TNF-α, IL-6, IL-1ß, and C5a were determined by ELISA. RESULTS: BCE exerted a confirmed but mild antipyretic effect on LPS-induced fever of rat. In vitro, it significantly lowered LPS-elevated TNF-α in the supernatant of rat complete blood cells and THP-1 cells, but failed to decrease IL-6 and IL-1ß. In murine endotoxemia models, BCE markedly decreased serum TNF-α, but had no impact on IL-6 and IL-1ß. BCE also restricted complement activation in vitro and in vivo. Nevertheless, the mixture of saikosaponin A and D could not suppress supernatant TNF-α of monocytes and serum TNF-α of endotoxemia mice. CONCLUSIONS: The present study dissects the peripheral mechanism for the antipyretic effect of BC used in the traditional way. Our findings indicate that BCE directly suppresses monocyte-produced TNF-α, thus decreasing circulating TNF-α, which may be responsible for its mild but confirmed antipyretic action.

Hyperthermia promotes degradation of the acute promyelocytic leukemia driver oncoprotein ZBTB16/RARα.

The acute promyelocytic leukemia (APL) driver ZBTB16/RARα is generated by the t(11;17) (q23;q21) chromosomal translocation, which is resistant to combined treatment of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) or conventional chemotherapy, resulting in extremely low survival rates. In the current study, we investigated the effects of hyperthermia on the oncogenic fusion ZBTB16/RARα protein to explore a potential therapeutic approach for this variant APL. We showed that Z/R fusion protein expressed in HeLa cells was resistant to ATO, ATRA, and conventional chemotherapeutic agents. However, mild hyperthermia (42 °C) rapidly destabilized the ZBTB16/RARα fusion protein expressed in HeLa, 293T, and OCI-AML3 cells, followed by robust ubiquitination and proteasomal degradation. In contrast, hyperthermia did not affect the normal (i.e., unfused) ZBTB16 and RARα proteins, suggesting a specific thermal sensitivity of the ZBTB16/RARα fusion protein. Importantly, we found that the destabilization of ZBTB16/RARα was the initial step for oncogenic fusion protein degradation by hyperthermia, which could be blocked by deletion of nuclear receptor corepressor (NCoR) binding sites or knockdown of NCoRs. Furthermore, SIAH2 was identified as the E3 ligase participating in hyperthermia-induced ubiquitination of ZBTB16/RARα. In short, these results demonstrate that hyperthermia could effectively destabilize and subsequently degrade the ZBTB16/RARα fusion protein in an NCoR-dependent manner, suggesting a thermal-based therapeutic strategy that may improve the outcome in refractory ZBTB16/RARα-driven APL patients in the clinic.

Resibufogenin, one of bufadienolides in toad venom, suppresses LPS-induced inflammation via inhibiting NF-κB and AP-1 pathways.

Toad venom is a traditional Chinese medicine that has a long history in treating infectious and inflammatory diseases, such as carbuncle, pharyngitis. As one of the major active components in toad venom, resibufogenin (RBG) possesses a variety of pharmacological activities, including lowering blood pressure, reducing proteinuria and preventing oxidative stress. But only its antitumor activity attracts widespread attention in these years. This study aimed to explore the nonnegligible anti-inflammatory activity of RBG in vivo and in vitro. In endotoxemia mice, a single intraperitoneal administration of RBG significantly lowered serum TNF-α, IL-6 and MCP-1 levels. In LPS-stimulated macrophages, RBG decreased LPS-induced pro-inflammatory mediators' productions (e.g., iNOS, IL-6, TNF-α and MCP-1) through suppressing their transcriptions. Mechanism study showed that RBG hindered IκBα phosphorylation and prevented nuclear translocation of p65, thus inactivating nuclear factor-κB (NF-κB) signaling. Concurrently, RBG also dampened activator protein-1 (AP-1) signaling through inhibiting the phosphorylation levels of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). Besides LPS (TLR4 ligand) model, RBG also inhibited Pam3CSK4 (TLR2 ligand)- or poly I:C (TLR3 ligand)-induced inflammatory reactions, suggesting that its target(s) site is(are) not on the cytomembrane. These findings not only support the pharmacological basis for the traditional use of toad venom in inflammatory diseases, but also provide a promising anti-inflammatory candidate.

Dietary supplementation with Weissella cibaria C-10 and Bacillus amyloliquefaciens T-5 enhance immunity against Aeromonas veronii infection in crucian carp (Carassiu auratus).

With the aim to discover novel lactic acid bacteria and Bacillus strains from fish as potential probiotics to replace antibiotics in aquaculture, the present study was conducted to isolate lactic acid bacteria and Bacillus from intestinal tract of healthy crucian carp (Carassiu auratus) and largemouth bass (Micropterus salmoides) and evaluate their resistance against Aeromonas veronii. Based on the evaluation of antibacterial activity and tolerance test, one strain of lactic acid bacteria (Weissella cibaria C-10) and one strain of Bacillus (Bacillus amyloliquefaciens T-5) with strong environmental stability were screened out. The safety evaluation showed that these two strains were non-toxic to crucian carp and were sensitive to most antibiotics. In vivo study, the crucian carps were fed a basal diet supplemented with W. cibaria C-10 (C-10), B. amyloliquefaciens T-5 (T-5) and W. cibaria C-10 + B. amyloliquefaciens T-5 (C-10+T-5), respectively, for 5 weeks. Then, various immune parameters were measured at 35 days of post-feeding. Results showed both probiotics could improve the activities of related immune enzymes, immune factors and non-specific immune antibodies in blood and organs (gill, gut, kidney, liver, and spleen) of crucian carp in varying degrees. Moreover, after 7 days of challenge experiment, the survival rates after challenged with A. veronii of W. cibaria C-10 (C-10), B. amyloliquefaciens T-5 (T-5) and W. cibaria C-10 + B. amyloliquefaciens T-5 (C-10+T-5) supplemented groups to the crucian carps were 20%, 33% and 22%, respectively. Overall, W. cibaria C-10 and B. amyloliquefaciens T-5 could be considered to be developed into microecological preparations for the alternatives of antibiotics in aquaculture.

Potentilla discolor ameliorates LPS-induced inflammatory responses through suppressing NF-κB and AP-1 pathways.

Potentilla discolor Bunge (PD) is a traditional Chinese medicine which has been widely used for the treatment of various inflammatory diseases (e.g., diarrhea, fever and furuncle). However, few studies focused on its effect on classical inflammation. This study aimed to investigate the anti-inflammatory effect and potential mechanism of the ethanol extract of the whole herbs of PD (EPD) in lipopolysaccharide (LPS)-induced inflammatory models. The obtained results showed that EPD decreased supernatant NO, tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) in LPS-activated RAW264.7 cells and mouse peritoneal macrophages. Moreover, its effect on NO was attributed to the suppression of iNOS expression rather than its activity. At the transcriptional level, EPD suppressed iNOS, TNF-α and MCP-1 mRNA expressions in LPS-stimulated RAW264.7 cells. Further study showed that EPD didn't affect the phosphorylation and degradation of IκBα, but yet impeded the nuclear translocation of p65 to inhibit NF-κB activation. Meanwhile, it also prevented JNK, ERK1/2 and p38 phosphorylation to dampen the activation of AP-1. In endotoxemia mouse model, EPD not only decreased interleukin-6, TNF-α and MCP-1 levels in serum, but also potently ameliorated diarrhea. These findings provide the theoretical basis for PD to treat inflammatory diseases, especially intestinal inflammation.

Forsythiasides-Rich Extract From Forsythiae Fructus Inhibits Mast Cell Degranulation by Enhancing Mitochondrial Ca2+ Uptake.

Mast cells (MCs) activated via IgE/FcεRI or MAS-related G protein coupled receptor (Mrgpr)-mediated pathway can release granules that play prominent roles in hypersensitivity reactions. Forsythiae Fructus, a well-known traditional Chinese medicine, has been clinically used for allergic diseases. Although previous studies indicated that Forsythiae Fructus extract inhibited compound 48/80-induced histamine release from MCs, its effect on IgE-dependent MC degranulation and possible underlying mechanisms remain to be explored. Herein, we prepared the forsythiasides-rich extract (FRE) and investigated its action on MC degranulation and explored its underlying mechanism. Our data showed that FRE could dampen IgE/FcεRI- and Mrgpr-mediated MC degranulation in vitro and in vivo. Mechanism study indicated that FRE decreased cytosolic Ca2+ (Ca2+ [c]) level rapidly and reversibly. Moreover, FRE decreased Ca2+ [c] of MCs independent of plasma membrane Ca2+-ATPase (PMCA), sarco/endoplasmic Ca2+-ATPase (SERCA) and Na+/Ca2+ exchanger (NCX). While, along with Ca2+ [c] decrease, the increase of mitochondrial Ca2+ (Ca2+ [m]) occurred simultaneously in FRE-treated RBL-2H3 cells. In the isolated mitochondria, FRE also promoted the subcellular organelle to uptake more extramitochondrial Ca2+. In conclusion, by increasing Ca2+ [m] uptake, FRE decreases Ca2+ [c] level to suppress MC degranulation. Our findings may provide theoretical support for the clinical application of Forsythiae Fructus on allergy and other MC-involved diseases.

Reversion mutation of cDNA CA8-204 minigene construct produces a truncated functional peptide that regulates calcium release in vitro and produces profound analgesia in vivo.

Intracellular calcium is critical in orchestrating neuronal excitability and analgesia. Carbonic anhydrase-8 (CA8) regulates intracellular calcium signaling through allosteric inhibition of neuronal inositol trisphosphate receptor 1 (ITPR1) to produce profound analgesia. Recently, we reported the "G" allele at rs6471859 represents cis-eQTL regulating alternative splicing of a 1697 bp transcript (CA8-204G) with a retained intron, alternative polyadenylation site and a new stop codon producing a functional 26 kDa peptide with an extended exon 3. In this study we show the reversion mutation (G to C) at rs6471859 within the CA8-204G expression vector also produced a stable 1697 bp transcript (CA8-204C) coding for a smaller peptide (~ 22 kDa) containing only the first three CA8 exons. Surprisingly, this peptide inhibited ITPR1 (pITPR1) activation, ITPR1-mediated calcium release in vitro; and produced profound analgesia in vivo. This is the first report showing CA8-204C codes for a functional peptide sufficient to regulate calcium signaling and produce profound analgesia.

HMQ-T-F5 (1-(4-(2-aminoquinazolin-7-yl)phenyl)-3-(2­bromo­5- (trifluoromethoxy)phenyl) thiourea) suppress proliferation and migration of human cervical HeLa cells via inhibiting Wnt/ß-catenin signaling pathway.

BACKGROUND: Drug therapy plays an important role in the treatment of cervical cancer, which is one of the most common solid tumors in women. Therefore, it is important to seek more effective and less toxic therapies. PURPOSE: The aim of this study is to investigate the therapeutic potential of HMQ-T-F5 (1-(4-(2-aminoquinazolin-7-yl)phenyl)-3-(2­bromo­5-(trifluoromethoxy) phenyl)thiourea) (F5) for cervical cancer and explore the related mechanism. METHODS: By performing MTT assay, colony formation assay, flow cytometry, wound-healing assay, transwell assay, immunofluorescent staining and siRNA assay, we study the effect of F5 on human cervical HeLa cells. The mechanism of F5 was also investigated. RESULTS: We found that F5 significantly inhibited HeLa cell proliferation, led to accumulation of cells in the S phase, and induced apoptosis and inhibited migration. Mechanistically, F5 inhibited HeLa cell growth and migration through repressing the expression and nuclear translocation of ß-catenin, enhancing Axin expression, inhibiting the phosphorylation of LRP5/6 and GSK3ß, as well as downregulating the Wnt downstream targeted proteins. Knockdown of a checkpoint ß-catenin by siRNA significantly attenuated HeLa cell proliferation. Furthermore, XAV939, an inhibitor of ß-catenin, was used to treat HeLa cells and the results demonstrated that F5 inhibited proliferation and migration via the inhibition of the Wnt/ß-catenin pathway. CONCLUSION: Our findings demonstrated that F5 can target ß-catenin potentially and is useful in the treatment of cervical cancer.

Wnt/ß-catenin signaling pathway is involved in regulating the migration by an effective natural compound brucine in LoVo cells.

BACKGROUND: Colorectal cancer remains the third most common malignancies and migration is one of the main factors for its high mortality rate. Brucine, a natural plant alkaloid, has been proved to possess a variety of pharmacological functions including anti-tumor activities. PURPOSE: The aim of this study was to investigate the inhibitory effect of brucine on the colorectal cancer and the underlying mechanism. METHODS: In this study, colony formation assay and transwell assay were used to investigate the effect of brucine on LoVo cells viability and migration. Immunofluorescence assay, western blot assay and Gelatin zymography assay were used to study the mechanism of brucine. Xenograft model in nude mice was induced to investigate the in vivo effect of brucine on LoVo cells. RESULTS: Brucine could significantly decrease the viability, inhibit the colony formation and induce the apoptosis of LoVo cells. Brucine could also suppress the migration of LoVo cells in a dose-dependent manner. Western blot analysis elucidated that the inhibition of migration was associated with the decreasing expression of matrix metalloproteinases including MMP2, MMP3 and MMP9. Moreover, we found that treatment of brucine could downregulate the expression of Frizzled-8, Wnt5a, APC and GSNK1A1, and increase the expression of AXIN1. Meanwhile, brucine also decreased the phosphorylation level of LRP5/6 and GSK3ß, and increased the level of p-ß-catenin. Xenografted model in nude mice study also revealed that oral administration of brucine could inhibit the growth and migration of LoVo cells by activating the expression of AXIN1 and p-ß-catenin. CONCLUSION: Brucine could suppress the migration of the colorectal cancer in vitro and in vivo and the effect was associated with the inhibition of the Wnt/ß-catenin signaling pathway.

Carbon monoxide attenuates lipopolysaccharide-induced lung injury by mitofusin proteins via p38 MAPK pathway.

BACKGROUND: The protective effects of carbon monoxide against the lipopolysaccharide (LPS)-induced lung injury were attributed to maintenance of mitochondrial dynamics, but the mechanisms remain unexplored. MATERIALS AND METHODS: Using a rat model of acute lung injury induced by LPS and the LPS attacking cell model, we investigated the effects of pretreatment of carbon monoxide molecule-2 (CORM-2) on the acute lung injury and expressions of mitofusin proteins that play a critical role in mitochondrial dynamics. RESULTS: We found that preadministration of CORM-2, not the inactive form of CORM-2, significantly reduced the lung injury, levels of inflammatory cytokines, and the degree of oxidative stress caused by LPS. What was more, it increased the expressions of mitofusin proteins. Similar findings were also found in LPS-stimulating cell model. However, when the cells were treated in combination with LPS, CORM-2, and SB203580, it completely abolished the protection of CORM-2, reflected by increased levels of inflammatory cytokines and malonaldehyde, decreased activities of superoxide dismutase, along with the lower expressions of mitofusin proteins and the ratio of p-p38 mitogen activated protein kinase to p38 mitogen activated protein kinase. CONCLUSIONS: Our observations suggest that pretreatment with CORM-2 could attenuate LPS-induced lung injury by inducing the expressions of mitofusin proteins via p38 mitogen activated protein kinase pathway.

Efficacy and tolerability of initial high vs low doses of S-(-)-amlodipine in hypertension.

In an 8-week randomized trial of patients with mild or moderate hypertension, the authors investigated the efficacy and tolerability of initial high (5.0 mg/d) vs low (2.5 mg/d) doses of S-(-)-amlodipine (equivalent to 5 and 10 mg of racemic amlodipine, respectively). In the S-(-)-amlodipine 2.5-mg group (n=263), 24-hour ambulatory systolic/diastolic blood pressure (±standard deviation) decreased from 131.5±15.0/82.1±10.7 mm Hg at baseline to 126.0±13.5/78.5±9.5 mm Hg at 8 weeks of follow-up by a least square mean (±standard error) change of 6.0±0.6/3.8±0.4 mm Hg. In the S-(-)-amlodipine 5-mg group (n=260), the corresponding changes were from 133.6±13.7/83.1±9.9 mm Hg to 125.0±12.0/78.2±8.9 mm Hg by 8.1±0.6/4.7±0.4 mm Hg, respectively. The between-group differences in changes in 24-hour systolic/diastolic blood pressure were 2.1/0.9 (P=.02/.17) mm Hg. Similar trends were observed for daytime and nighttime ambulatory and clinic blood pressure. The incidence rate was similar for all adverse events. An initial high dose of S-(-)-amlodipine improved ambulatory blood pressure control with similar tolerability as an initial low dose in hypertension.

Isolation of active compounds from methanol extracts of Toddalia asiatica against Ichthyophthirius multifiliis in goldfish (Carassius auratus).

The parasitic ciliate Ichthyophthirius multifiliis infests all species of freshwater fish and can cause severe economic losses in fish breeding. The present study aims to evaluate the antiparasitic activity of the active components from Toddalia asiatica against I. multifiliis. Bioassay-guided fractionation and isolation of compounds with antiparasitic activity were performed on the methanol extract of T. asiatica yielding two bioactive compounds: chelerythrine and chloroxylonine identified by comparing spectral data (NMR and ESI-MS) with literature values. Results from in vitro antiparasitic assays revealed that chelerythrine and chloroxylonine could be 100% effective against I. multifiliis at the concentration of 1.2 mg L(-1) and 3.5 mg L(-1), with the median effective concentration (EC50) values of 0.55 mg L(-1) and 1.90 mg L(-1) respectively. In vivo experiments demonstrated that fish treated with chelerythrine and chloroxylonine at the concentrations of 1.8 and 8.0 mg L(-1) carried significantly fewer parasites than the control (P<0.05). The acute toxicity (LC50) of chelerythrine for goldfish was 3.3 mg L(-1).

Transcutaneous electrical nerve stimulation on acupoints relieves labor pain: a non-randomized controlled study.

OBJECTIVE: To investigate the efficacy of transcutaneous electrical nerve stimulation (TENS) on four specific acupuncture points Hegu (LI4), Neiguan (PC6), Danshu (BL19) and Weishu (BL21) for reducing pain in labor. METHODS: A total of 160 voluntary nulliparous women who were willing to receive TENS for analgesia were assigned to the treatment group after cervical dilation of more than 2 cm. Another 145 matched nullipara were recruited as the control group. Visual analogue scale (VAS) was used to assess the pain before and 0.5 h after the application of TENS. Then, VAS was assessed every one hour until delivery. Percentage of VAS score decreased by > 25% was the primary outcome, the delivery mode and neonatal outcome were measured as secondary outcomes. Adverse reactions were also recorded during TENS. RESULTS: The percentage of VAS score decreased by > 25% was 68.6% in the TENS treatment group. Maternal delivery mode and neonatal outcomes were not significantly different between the two groups. In addition, the incidence of postpartum hemorrhage in the TENS treatment group was less than the control group (P<0.05). There was no adverse reaction recorded with TENS on acupoints. CONCLUSION: As a novel and non-invasive approach, TENS on specific acupoints including Hegu (LI4), Neiguan (PC6), Danshu (BL19) and Weishu (BL21) was an effective method for analgesia in labor.

[Influence of Mn2+ on the biotechmycin fermentation].

The effect of Mn2+ on the biotechmycin fermentation by Bioengineered strain WSJ-l-195 was studied. In the fermentation process, Mn2+ could improve the biological potency significantly, especially when Mn2+ concentration was 5 mmol/L added at 24 h. The pH profile of fermentation broth decreased gradually after 5 mmol/L Mn2+ supplemented at 24 h, and PMV was lower than that of the control sample. Further research about the influence of Mn2+ on the biosynthesis of biotechmycin was carried out in the aspect of organic acids. The results showed that concentrations of organic acids in a fermentation with 5 mmol/L Mn2+ supplemented at 24 h had been changed greatly, especially the concentration of propionic acid, of which the highest value was about 6 times as that in the control sample at 84 h. In addition, it was found that the yield of biotechmycin could be improved significantly with tiny amount of propionic acid added. Therefore, it can be concluded that Mn2+ has profound influence on the biosynthesis of biotechmycin: it enriches the biotechmycin precursor pool such as propionic acid and thus improves the yield of biotechmycin.

A novel PEPP homeobox gene, TOX, is highly glutamic acid rich and specifically expressed in murine testis and ovary.

The homeobox gene superfamily has been highly conserved throughout evolution. These genes act as transcription factors during several important developmental processes. To explore the functional roles of homeobox genes in spermatogenesis, we performed a degenerate oligonucleotide polymerase chain reaction (PCR) screening of a testis cDNA library and isolated a novel mouse homeobox gene. This gene, which we named Tox, encodes a homeodomain protein distantly related to members of the Paired/Pax (Prd/Pax) family. A phylogenetic analysis revealed Tox to be a member of the recently defined PEPP subfamily of Paired-like homeobox genes. Tox was mapped to chromosome X, with its homeodomain organized into three exons. A special feature of Tox is that the encoded protein sequence contains two poly-glutamic acid (poly E) stretches, which make Tox highly acidic. Tox transcripts were detected predominately in the testis and ovary of mice. Tox expression in testes was initiated soon after birth, mainly in Sertoli cells and spermatogonia; however, in adult mice, Tox expression shifts to the spermatids and spermatozoa. Tox expression in ovaries was detected in somatic cells of follicles, early on in theca cells, and in both granulosa and theca cells at the later stages of follicular development. Based on these results, Tox may play an important role during gametogenesis.