Manual acupuncture ameliorates inflammatory pain by upregulating adenosine A3 receptor in complete Freund's adjuvant-induced arthritis rats.

BACKGROUND: Adenosine A3 receptor (A3R) exerts analgesic, anti-inflammatory, and anti-nociceptive effects. In this study, we determined the analgesic mechanism of manual acupuncture (MA) in rats with complete Freund's adjuvant (CFA)-induced arthritis and explored whether MA ameliorates inflammation in these rats by upregulating A3R. METHODS: Sixty Sprague Dawley (SD) rats were randomly divided into the following groups: Control, CFA, CFA + MA, CFA + sham MA, CFA + MA + DMSO, CFA + MA + IB-MECA, and CFA + MA + Reversine groups. The arthritis rat model was induced by injecting CFA into the left ankle joints. Thereafter, the rats were subjected to MA (ST36 acupoint) for 3 days. The clinical indicators paw withdrawal latency (PWL), paw withdrawal threshold (PWT), and open field test (OFT) were used to determine the analgesic effect of MA. In addition, to explore the effect of A3R on inflammation after subjecting arthritis rats to MA, IB-MECA (A3R agonist) and Reversine (A3R antagonist) were injected into ST36 before MA. RESULTS: MA ameliorated the pathological symptoms of CFA-induced arthritis, including the pain indicators PWL and PWT, number of rearing, total ambulatory distance, and activity trajectory. Furthermore, after MA, the mRNA and protein expression of A3R was upregulated in CFA-induced arthritis rats. In contrast, the protein levels of TNF-α, IL-1ß, Rap1, and p-p65 were downregulated after MA. Interestingly, the A3R agonist and antagonist further downregulated and upregulated inflammatory cytokine expression, respectively, after MA. Furthermore, the A3R antagonist increased the degree of ankle swelling after MA. CONCLUSION: MA can alleviate inflammatory pain by inhibiting the NF-κB signaling pathway via upregulating A3R expression of the superficial fascia of the ST36 acupoint site in CFA-induced arthritis rats.

Aloperine alleviates lipopolysaccharide-induced acute lung injury by inhibiting NLRP3 inflammasome activation.

RATIONALE: Excessive activation of the NLRP3 inflammasome is involved in the pathological progression of acute lung injury (ALI). Aloperine (Alo) has anti-inflammatory effects in many inflammatory disease models; however, its role in ALI remains elusive. In this study, we addressed the role of Alo in NLRP3 inflammasome activation in both ALI mice and LPS-treated RAW264.7 cells. METHODS: The activation of the NLRP3 inflammasome in LPS-induced ALI lungs was investigated in C57BL/6 mice. Alo was administered in order to study its effect on NLRP3 inflammasome activation in ALI. RAW264.7 cells were used to evaluate the underlying mechanism of Alo in the activation of the NLRP3 inflammasome in vitro. RESULTS: The activation of the NLRP3 inflammasome occurs in the lungs and RAW264.7 cells under LPS stress. Alo attenuated the pathological injury of lung tissue as well as downregulates the mRNA expression of NLRP3 and pro-caspase-1 in ALI mice and LPS-stressed RAW264.7 cells. The expression of NLRP3, pro-caspase-1, and caspase-1 p10 were also significantly suppressed by Alo in vivo and in vitro. Furthermore, Alo decreased IL-1ß and IL-18 release in ALI mice and LPS-induced RAW264.7 cells. In addition, ML385, a Nrf2 inhibitor, weakened the activity of Alo, which inhibited the activation of the NLRP3 inflammasome in vitro. CONCLUSION: Alo reduces NLRP3 inflammasome activation via the Nrf2 pathway in ALI mice.

DEFB126 polymorphisms and association with idiopathic asthenozoospermia in China.

Idiopathic asthenozoospermia, a common factor in male infertility, is characterized by altered sperm motility function in fresh ejaculate. Although the ß-defensin 126 (DEFB126) protein is associated with asthenozoospermia, DEFB126 gene polymorphisms have not been extensively studied. Therefore, the association between DEFB126 gene polymorphisms and asthenozoospermia requires further investigation. Screening was performed by semen analysis, karyotype analysis, and Y microdeletion detection, and 102 fertile men and 106 men with asthenozoospermia in Chengdu, China, were selected for DEFB126 gene sequence analyses. Seven nucleotide mutations and two nucleotide deletions in the DEFB126 gene were detected. rs11467417 (317-318 del/del), rs11467497 (163-166 wt/del), c.152T>C, and c.227A>G were significantly different between the control and asthenozoospermia groups, likely representing high-risk genetic factors for asthenozoospermia among males. DEFB126 expression was not observed in sperm with rs11467497 homozygous deletion and was unstable in sperm with rs11467417 homozygous deletion. The rs11467497 four-nucleotide deletion leads to truncation of DEFB126 at the carboxy-terminus, and the rs11467417 binucleotide deletion produces a non-stop messenger RNA (mRNA). The above deletions may be responsible for male hypofertility and infertility by reducing DEFB126 affinity to sperm surfaces. Based on in silico analysis, the amino acids 51M and 76K are located in the highly conserved domain; c.152T>C (M51T) and c.227A>G (K76R) are predicted to be damaging and capable of changing alternative splice, structural and posttranslational modification sites of the RNA, as well as the secondary structure, structural stability, and hydrophobicity of the protein, suggesting that these mutations are associated with asthenozoospermia.

Beneficial effects of aloperine on inflammation and oxidative stress by suppressing necroptosis in lipopolysaccharide-induced acute lung injury mouse model.

RATIONALE: Alveolar epithelial cell death, inflammation, and oxidative stress are typical features of acute lung injury (ALI). Aloperine (Alo), an alkaloid isolated from Sophora alopecuroides, has been reported to display various biological effects, such as anti-inflammatory, immunoregulatory, and anti-oxidant properties. In this study, we investigated the effects and mechanisms of Alo in treating a lipopolysaccharide (LPS)-induced ALI in a murine model. METHODS: The effects of Alo in LPS-induced ALI were investigated in C57BL/6 mice. The RIPK1 inhibitor (Nec-1) and the RIPK3 inhibitor (GSK'872) were used to evaluate the relationship of necroptosis, NF-κB activation, and PDC subunits in LPS-treated mouse alveolar epithelial cells (MLE-12). Then the effects of Alo on necroptosis, inflammation, and oxidative stress of LPS-stimulated MLE-12 cells were evaluated. RESULTS: Alo significantly attenuated histopathological lung injuries and reduced lung wet/dry ratio in LPS-induced ALI mice. Alo also remarkedly reduced total protein and neutrophils recruitment in bronchoalveolar lavage fluid of ALI mice. Meanwhile, Alo ameliorated the LPS-induced necroptosis in the lungs of ALI mice. The RIPK3 inhibitor GSK'872, but not the RIPK1 inhibitor Nec-1, reversed LPS-induced p65 phosphorylation and translocation to the nucleus in MLE-12 cells. GSK'872 also reversed the LPS-induced increase in ROS and binding of RIPK3 and PDC subunits in MLE-12 cells. Moreover, Alo down-regulated the levels of p-RIPK1, p-RIPK3, p-MLKL, p-p65, the translocation of p65 to the nucleus, and reduced the expression of IL-6 and IL-8 in LPS-stimulated MLE-12 cells. Alo also inhibited the binding of RIPK3 and PDC-E1α, PDC-E1ß, PDC-E2, and PDC-E3 and the ROS production in LPS-treated MLE-12 cells. CONCLUSION: The present study validated the beneficial effects of Alo on LPS-induced ALI , suggesting Alo may be a new drug candidate against ALI.

Phage peptides mediate precision base editing with focused targeting window.

Base editors (BEs) are genome engineering tools that can generate nucleotide substitutions without introducing double-stranded breaks (DSBs). A variety of strategies have been developed to improve the targeting scope and window of BEs. In a previous study, we found that a bacteriophage-derived peptide, referred to as G8PPD, could improve the specificity of Cas9 nuclease. Herein, we investigate the applicability of G8PPD as molecular modulators of BEs. We show that G8PPD can improve cytidine base editor (CBEs) and adenine base editor (ABE) to more focused targeting windows. Notably, in a cell-based disease model, G8PPD increases the percentage of perfectly edited gene alleles by BEs from less than 4% to more than 38% of the whole population. In addition, G8PPD can improve the targeting scope of BE in mouse embryos. In summary, our study presents the peptidyl modulators that can improve BEs for precision base editing.

KPT330 improves Cas9 precision genome- and base-editing by selectively regulating mRNA nuclear export.

CRISPR-based genome engineering tools are associated with off-target effects that constitutively active Cas9 protein may instigate. Previous studies have revealed the feasibility of modulating Cas9-based genome- and base-editing tools using protein or small-molecule CRISPR inhibitors. Here we screened a set of small molecule compounds with irreversible warhead, aiming to identifying small-molecule modulators of CRISPR-Cas9. It was found that selective inhibitors of nuclear export (SINEs) could efficiently inhibit the cellular activity of Cas9 in the form of genome-, base- and prime-editing tools. Interestingly, SINEs did not function as direct inhibitors to Cas9, but modulated Cas9 activities by interfering with the nuclear export process of Cas9 mRNA. Thus, to the best of our knowledge, SINEs represent the first reported indirect, irreversible inhibitors of CRISPR-Cas9. Most importantly, an FDA-approved anticancer drug KPT330, along with other examined SINEs, could improve the specificities of CRISPR-Cas9-based genome- and base editing tools in human cells. Our study expands the toolbox of CRISPR modulating elements and provides a feasible approach to improving the specificity of CRISPR-Cas9-based genome engineering tools.

Allosteric inhibition of CRISPR-Cas9 by bacteriophage-derived peptides.

BACKGROUND: CRISPR-Cas9 has been developed as a therapeutic agent for various infectious and genetic diseases. In many clinically relevant applications, constitutively active CRISPR-Cas9 is delivered into human cells without a temporal control system. Excessive and prolonged expression of CRISPR-Cas9 can lead to elevated off-target cleavage. The need for modulating CRISPR-Cas9 activity over time and dose has created the demand of developing CRISPR-Cas off switches. Protein and small molecule-based CRISPR-Cas inhibitors have been reported in previous studies. RESULTS: We report the discovery of Cas9-inhibiting peptides from inoviridae bacteriophages. These peptides, derived from the periplasmic domain of phage major coat protein G8P (G8PPD), can inhibit the in vitro activity of Streptococcus pyogenes Cas9 (SpCas9) proteins in an allosteric manner. Importantly, the inhibitory activity of G8PPD on SpCas9 is dependent on the order of guide RNA addition. Ectopic expression of full-length G8P (G8PFL) or G8PPD in human cells can inactivate the genome-editing activity of SpyCas9 with minimum alterations of the mutation patterns. Furthermore, unlike the anti-CRISPR protein AcrII4A that completely abolishes the cellular activity of CRISPR-Cas9, G8P co-transfection can reduce the off-target activity of co-transfected SpCas9 while retaining its on-target activity. CONCLUSION: G8Ps discovered in the current study represent the first anti-CRISPR peptides that can allosterically inactivate CRISPR-Cas9. This finding may provide insights into developing next-generation CRISPR-Cas inhibitors for precision genome engineering.

AXL promotes Zika virus infection in astrocytes by antagonizing type I interferon signalling.

Zika virus (ZIKV) is associated with neonatal microcephaly and Guillain-Barré syndrome1,2. While progress has been made in understanding the causal link between ZIKV infection and microcephaly3-9, the life cycle and pathogenesis of ZIKV are less well understood. In particular, there are conflicting reports on the role of AXL, a TAM family kinase receptor that was initially described as the entry receptor for ZIKV10-22. Here, we show that while genetic ablation of AXL protected primary human astrocytes and astrocytoma cell lines from ZIKV infection, AXL knockout did not block the entry of ZIKV. We found, instead, that the presence of AXL attenuated the ZIKV-induced activation of type I interferon (IFN) signalling genes, including several type I IFNs and IFN-stimulating genes. Knocking out type I IFN receptor α chain (IFNAR1) restored the vulnerability of AXL knockout astrocytes to ZIKV infection. Further experiments suggested that AXL regulates the expression of SOCS1, a known type I IFN signalling suppressor, in a STAT1/STAT2-dependent manner. Collectively, our results demonstrate that AXL is unlikely to function as an entry receptor for ZIKV and may instead promote ZIKV infection in human astrocytes by antagonizing type I IFN signalling.

Complex molecular mechanisms underlying seedling salt tolerance in rice revealed by comparative transcriptome and metabolomic profiling.

To understand the physiological and molecular mechanisms underlying seedling salt tolerance in rice (Oryza sativa L.), the phenotypic, metabolic, and transcriptome responses of two related rice genotypes, IR64 and PL177, with contrasting salt tolerance were characterized under salt stress and salt+abscisic acid (ABA) conditions. PL177 showed significantly less salt damage, lower Na(+)/K(+) ratios in shoots, and Na(+) translocation from roots to shoots, attributed largely to better salt exclusion from its roots and salt compartmentation of its shoots. Exogenous ABA was able to enhance the salt tolerance of IR64 by selectively decreasing accumulation of Na(+) in its roots and increasing K(+) in its shoots. Salt stress induced general and organ-specific increases of many primary metabolites in both rice genotypes, with strong accumulation of several sugars plus proline in shoots and allantoin in roots. This was due primarily to ABA-mediated repression of genes for degradation of these metabolites under salt. In PL177, salt specifically up-regulated genes involved in several pathways underlying salt tolerance, including ABA-mediated cellular lipid and fatty acid metabolic processes and cytoplasmic transport, sequestration by vacuoles, detoxification and cell-wall remodeling in shoots, and oxidation-reduction reactions in roots. Combined genetic and transcriptomic evidence shortlisted relatively few candidate genes for improved salt tolerance in PL177.

SS1 (NAL1)- and SS2-Mediated Genetic Networks Underlying Source-Sink and Yield Traits in Rice (Oryza sativa L.).

Source leaf/sink capacity (SS) traits are important determinants of grain yield (GY) of rice. To understand the genetic basis of the SS relationship in rice, five SS and GY traits of rice were genetically dissected using two reciprocal introgression populations. Seventy-three QTL affecting the SS and GY traits were identified, most of which were detected in one of the parental genetic backgrounds (GBs). Two major QTL at bins 4.7 (SS1) and 3.12 (SS2) were associated consistently with all measured SS and yield traits in both GBs across two contrasting environments. Strong interactions between SS1/SS2 and the detected QTL led us to the discovery of genetic networks affecting the SS and GY traits. The SS1 acted as a regulator controlling two groups of downstream QTL affecting the source leaf width and grain number per panicle (GNP). SS2 functioned as a regulator positively regulating different groups of downstream QTL affecting the source leaf length, GNP, grain weight, and GY. Map-based cloning of SS1 indicates that SS1 is NAL1 involved in polar auxin/IAA transport. Different alleles at NAL1 were apparently able to qualitatively and/or quantitatively control the IAA transport from the apical meristem to different plant tissues and thus regulate those downstream loci/pathways controlling different SS traits of rice. There was a functional allele and a non-functional mutation in the parents at each of the QTL downstream of SS1 or SS2, which were detectable only in the presence of the functional allele of SS1 or SS2. Our results provided direct evidence that SS and yield traits in rice are controlled by complex signaling pathways and suggest further improvement of rice yield potential with enhanced and balanced SS relationships can be achieved by accurately manipulating allelic combinations at loci in the SS1 and SS2 mediated pathways.

Role of CREB in vasoactive intestinal peptide-mediated wound healing in human bronchial epithelial cells.

Vasoactive intestinal peptide (VIP) is one of the most important sensory neuropeptides in respiratory system. We previously reported that VIP enhances wound healing and proliferation of human bronchial epithelial cells (HBECs), and these effects are mediated by intracellular signaling molecules such as protein kinase A (PKA), protein kinase C (PKC), Calmodulin (CaM), and extracellular signal-regulated kinase (ERK). In the present study, we further investigated the role of cAMP Response Element Binding Protein (CREB) in VIP-promoted protective effects in mechanical-damaged HBECs. VIP-mediated wound healing and cell proliferation in HBECs was inhibited by CREB antisense oligonucleotides (ASO) in a time-dependent manner. VIP increased the CREB DNA binding activity and expression of the p-CREB that were inhibited by VIP receptor antagonist. Increased CREB DNA binding activity and expression of the p-CREB were also partially inhibited by PKA and ERK inhibitors. These results suggest that the VIP-mediated wound repair of HBECs is associated with activation of CREB via PKA and ERK dependent pathway.

Ozone stress down-regulates the expression of cystic fibrosis transmembrane conductance regulator in human bronchial epithelial cells.

To investigate abnormalities of cystic fibrosis transmembrane conductance regulator (CFTR) expression in chronic inflammatory airway diseases and its regulation mechanisms, the present study was designed to observe the expression of CFTR, CFTR chloride current and the possible relevant signal pathways in in vitro and in vivo bronchial epithelium by using real-time PCR, immunofluorescence, Western blot and whole cell patch-clamp. The results demonstrated that CFTR staining was decreased in rat airway epithelium under ozone stress. Ozone stress also down-regulated CFTR protein and mRNA expression and CFTR chloride current in cultured human bronchial epithelial cells (HBEC). STAT1 signal pathway was checked to investigate the signal mechanism. It was found that pretreatment with STAT1 inhibitor attenuated the down-regulated CFTR expression induced by ozone stress. We also observed that ozone stress accelerated the phosphorylation of STAT1 in HBEC, which could be influenced by some signaling molecules related to the early transduction of cellular stress. Furthermore, reactive oxygen species inhibitors N-acetylcysteine and nitric oxide synthase inhibitor aminoguanidine increased the expression of CFTR. Ozone stress could down-regulate the expression of CFTR and decrease CFTR chloride current in HBEC. The signal mechanism which referred to cascade events in cells included early oxidative stress signal transmission molecules, and subsequently transcription modulator STAT1.

[Effect of calcitonin-gene-related peptide on MMP-9 production in human bronchial epithelial cells].

OBJECTIVE: To examine the expression of matrix metalloproteinase-9 (MMP-9) in human bronchial epithelial cells treated with calcitonin-gene-related peptide (CGRP). METHODS: RT-PCR and gelatin zymography were performed to examine the dynamic expression and activity of MMP-9 in human bronchial epithelial cells at different doses (10(-10), 10(-9), 10(-8), 10(-7), and 10(-6)mol/L) and different time points (6,12,18,24,36, and 48h) after the stimulation of CGRP. RESULTS: The unstimulated human bronchial epithelial cells only secreted a small amount of MMP-9. After the CGRP stimulation, the expression of MMP-9 presented in a concentration-dependent (10(-10), 10(-9), 10(-8), 10(-7), and 10(-6) mol/L) and time-dependent (6,12,18,24,36, and 48 h) manners (P<0.01) in human bronchial epithelial cells. The effect of CGRP could be diminished by H-7 and W-7, an antagonist of protein kinase C (PKC) and calmodulin (CaM) (P<0.05). CONCLUSION: CGRP can stimulate the secretion and expression of MMP-9 in human bronchial epithelial cells, and the signal transduction is partly via the PKC and CaM pathway.

Intracellular signaling molecules involved in vasoactive intestinal peptide-mediated wound healing in human bronchial epithelial cells.

Vasoactive intestinal peptide (VIP), a non-adrenergic, non-cholinergic neuromediator, plays an important role in maintaining the bronchial tone of the airway and has anti-inflammatory properties. Recently, we reported that VIP enhances wound repair in human bronchial epithelial cells (HBEC). In the present study, we have identified the intracellular signaling molecules that are involved in VIP-mediated wound healing in HBEC. The effects of VIP on wound repair of HBEC were partially blocked by H-7 (a protein kinase C (PKC) inhibitor), W-7 (a calmodulin inhibitor), H-89 (a protein kinase A (PKA) inhibitor), and PD98059 (a specific extracellular signal-regulated kinase (ERK) inhibitor). VIP-induced chemotactic migration was inhibited in the presence of W-7, H-89, PD98059 or H-7. H-7, W-7, and H-89 were also found to decrease VIP-induced expression of Ki67 as well as the proliferation index in HBEC. Furthermore, H-7, W-7, H-89, and PD98059 inhibited the expression of E-cd protein and mRNA induced by VIP. These results suggest that intracellular signaling molecules such as PKA, PKC, ERK, and calmodulin play important role in VIP-mediated wound healing of HBEC.

[Effects of CGRP on LPS-induced MMP-9 secretion by alveolar macrophages].

AIM: To explore the effects of calcitonin-gene-related peptide (CGRP) on LPS-induced MMP-9 secretion by alveolar macrophages (AM) in vitro. METHODS: The supernatant of LPS-induced Wistar rat AM from different intervention groups were collected to measure the activity by gelatin zymography. RESULTS: (Only secreting a small amount of MMP-9 with unstimulated AM, LPS stimulated MMP-9 production in a concentration-dependent manner (p < 0.01). (2) The activity of MMP-9 in CGRP intervention groups at different levels were significantly lower than those in non-intervention group (p < 0.01). (3) The inhibiting effects of CGRP were diminished by H-7 and W-7, an antagonist of protein kinase C (PKC) and calmodulin (CaM) (p < 0.05). CONCLUSION: These data suggested that CGRP involved in the MMP-9 secretion by AM, partly, via PKC and CaM pathway.

Vasoactive intestinal peptide enhances wound healing and proliferation of human bronchial epithelial cells.

In the present study, we investigated the effects of vasoactive intestinal peptide (VIP) on wound healing of bronchial epithelium. Wound healing of the mechanical damaged human bronchial epithelial cells (HBEC) was observed in the absence or presence of VIP. Effects of VIP on chemotactic migration, cell proliferation of HBEC were also tested. HBEC chemotaxis was assessed by the blind well chamber technique, the cell cycle was determined by flow cytometry, and cell proliferation was determined by measuring the expression of proliferating cell nuclear antigen Ki67. Effects of VIP on epithelial E-cadherins protein and mRNA were also measured by immunohistochemistry and RT-PCR. The results showed that VIP accelerated the recovery of wound area of HBEC. VIP increased the migration and proliferation of HBEC, and these effects were blocked by a VPAC1 receptor antagonist. VIP also increased the expression of E-cadherin mRNA and protein in HBEC, suggesting that protective effects of VIP on wound healing may be related to its ability to increase the expression of E-cadherin. In conclusion, VIP has protective effects against human bronchial epithelial cell damage, and the beneficial effects of VIP might be mediated, at least in part, by VPAC1, and associated with increased expression of E-cadherin.