Objective Ticks serve as vectors for transmitting Babesia microti.However,the specific mechanism remains unclear.This study aimed to investigate the effect of tick autophagy molecules on the proliferation of Babesia microti.Methods An experimental model of infected and uninfected mice was used to collect tick materials for proteomic analysis to identify differentially expressed autophagy-related molecules in Haemaphysalis longicornis.The cloning of the HlATG8 gene,protein expression,and production of polyclonal antibodies were completed.The HlATG8 gene was then knocked down using RNAi interference technology.Results The tick autophagy molecule,HlATG8,was identified and found to be significantly upregulated in ticks infected with Babesia microti.The load of Babesia microti in ticks increased significantly following the knockdown of the HlATG8 gene.Conclusions The tick autophagy molecule in Hae.longicornis,HlATG8,inhibits the proliferation of Babesia.

Objective:To investigate the role of microRNA-709(miR-709)in erythroid development and its correlation with multiple hematological diseases.Methods:The expression of miR-709 in multiple tissues of mice was detected by qRT-PCR;The expression of miR-709 and other miRNAs in day-14.5 fetal liver cells from mouse embryos was detected by transcriptome microarray analysis;The expression of miR-709 in nucleated red blood cells derived from bone marrow and spleen,and in peripheral blood erythrocytes of mice was detected by magnetic bead sorting combined with qRT-PCR;The expression of miR-709 during erythroid differentiation of murine erythroleukemia(MEL)cells was analyzed by cell culture and qRT-PCR;The expression of miR-709 during erythroid lineage differentiation of mouse erythroid precursor cells derived from fetal livers was analyzed by magnetic bead sorting,flow cytometry,cell culture and qRT-PCR;The expression of miR-709 in peripheral blood of patients with different hematological diseases was measured by qRT-PCR.Results:Among the various tissues examined in mice,miR-709 exhibited the highest expression in peripheral blood,followed by high expression levels in muscle,bone marrow,and liver;In day-14.5 fetal liver cells from mouse embryos,miR-709 was highly expressed,significantly surpassing miR-451,which was most highly expressed in mature red blood cells;In nucleated red blood cells derived from mouse bone marrow and spleen,the expression of miR-709 was higher than that of miR-451,whereas the opposite pattern was observed in peripheral blood;During the differentiation of erythroid precursor cells derived from mouse embryonic liver,the expression level of miR-709 first increased and then decreased;During the differentiation of MEL cells,the expression of miR-709 gradually increased;Compared with the healthy controls,patients with myelodysplastic syndrome(MDS),α-thalassemia and β-thalassemia expressed lower levels of miR-709 in peripheral blood;while the expression of miR-709 in patients with infectious hemolytic anemia(IHA)was higher than that in healthy controls.Conclusion:miR-709 is highly expressed in the early stage of erythropoiesis and exhibits dynamic changes during erythroid development,potentially playing an important role.It is also differentially expressed in different hematological diseases,which is expected to serve as a promising biomarker and therapeutic target in the clinical diagnosis and treatment of hematological diseases.

Objective:To investigate the role of microRNA-709(miR-709)in erythroid development and its correlation with multiple hematological diseases.Methods:The expression of miR-709 in multiple tissues of mice was detected by qRT-PCR;The expression of miR-709 and other miRNAs in day-14.5 fetal liver cells from mouse embryos was detected by transcriptome microarray analysis;The expression of miR-709 in nucleated red blood cells derived from bone marrow and spleen,and in peripheral blood erythrocytes of mice was detected by magnetic bead sorting combined with qRT-PCR;The expression of miR-709 during erythroid differentiation of murine erythroleukemia(MEL)cells was analyzed by cell culture and qRT-PCR;The expression of miR-709 during erythroid lineage differentiation of mouse erythroid precursor cells derived from fetal livers was analyzed by magnetic bead sorting,flow cytometry,cell culture and qRT-PCR;The expression of miR-709 in peripheral blood of patients with different hematological diseases was measured by qRT-PCR.Results:Among the various tissues examined in mice,miR-709 exhibited the highest expression in peripheral blood,followed by high expression levels in muscle,bone marrow,and liver;In day-14.5 fetal liver cells from mouse embryos,miR-709 was highly expressed,significantly surpassing miR-451,which was most highly expressed in mature red blood cells;In nucleated red blood cells derived from mouse bone marrow and spleen,the expression of miR-709 was higher than that of miR-451,whereas the opposite pattern was observed in peripheral blood;During the differentiation of erythroid precursor cells derived from mouse embryonic liver,the expression level of miR-709 first increased and then decreased;During the differentiation of MEL cells,the expression of miR-709 gradually increased;Compared with the healthy controls,patients with myelodysplastic syndrome(MDS),α-thalassemia and β-thalassemia expressed lower levels of miR-709 in peripheral blood;while the expression of miR-709 in patients with infectious hemolytic anemia(IHA)was higher than that in healthy controls.Conclusion:miR-709 is highly expressed in the early stage of erythropoiesis and exhibits dynamic changes during erythroid development,potentially playing an important role.It is also differentially expressed in different hematological diseases,which is expected to serve as a promising biomarker and therapeutic target in the clinical diagnosis and treatment of hematological diseases.

AIM To investigate the renal protective effects of Shiwei Ruxiang Powder on gouty nephritis in rats based on mitophagy.METHODS Rats were randomly divided into the blank group,the model group,the low-dose,medium-dose,and high-dose Shiwei Ruxiang Powder groups(200,400,800 mg/kg)and allopurinol group(10 mg/kg).The rat model of gouty nephropathy was established by gavage of potassium oxyzinate(750 mg/kg)and uric acid(300 mg/kg).The rats had their levels of UA,SCr,BUN,XOD,SOD,MDA,ROS measured by automatic biochemical analyzer,ELISA and chemical fluorescence method;their renal pathological changes observed by HE staining;their apoptosis of renal tissue cells observed by TUNEL staining;and their mRNA and protein expressions of IL-1β,TNF-α,Bax,Bcl-2,caspase-3,caspase-9,PINK1,Parkin and LC3-Ⅱ detected by RT-qPCR and Western blot.RESULTS Compared with the model group,Shiwei Ruxiang Powder groups displayed dose-dependently decreased serum levels of UA,BUN and SCr,renal deposition of urate crystal and apoptosis(P<0.05);decreased renal levels of ROS and inflammatory factors IL-1β and TNF-α(P<0.05);and increased renal expressions of mitochondrial autophagy-related proteins PINK1,Parkin and LC3-Ⅱ(P<0.01).CONCLUSION Shiwei Ruxiang Powder may relieve gouty kidney injury in rats by reducing the uric acid level,the renal oxidative stress and inflammatory response,and activating mitophagy pathway as well.

This study rapidly identified and quantified the chemical components of the Wuzhuyu Decoction nanophase(WZYD-N) and suspension phase(WZYD-S) using ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry(UPLC-QQQ-MS/MS). Based on preliminary pharmacodynamic experiments and network pharmacology analysis, the differential anti-inflammatory and analgesic activities of WZYD-N and WZYD-S were explored to understand their pharmacodynamic basis. WZYD-N and WZYD-S were separated by differential centrifugation-dialysis, and their particle size, Zeta potential, PDI, and morphology were characterized by dynamic light scattering and transmission electron microscopy. A method was established to quantify 23 representative components of WZYD using UPLC-QQQ-MS/MS, clarifying the differences in component content between the two phases. The anti-inflammatory and analgesic activities of WZYD-N and WZYD-S were preliminarily investigated using the acetic acid-induced enhanced capillary permeability inflammation model and the acetic acid writhing pain model. Network pharmacology was applied to screen the key anti-inflammatory and analgesic targets and active components of WZYD, and the relationship between the components and pharmacodynamics of WZYD-N and WZYD-S was analyzed based on quantitative results. The results showed that WZYD-N primarily consisted of spherical self-assembled aggregates around 200 nm, with a PDI of approximately 0.299 and a zeta potential of-22.1 mV. With an equivalent amount of crude drugs, obacunone and dihydroevocarpine were quantified in equal amounts in WZYD-N and WZYD-S. The content of rutaecarpine, evocarpine, rutaevine, limonin, and ginsenoside Ro was higher in WZYD-S, while 15 other components, including evodiamine, dehydroevodiamine, ginsenoside Re, 6-gingerol, and ginsenoside Rg_1, were higher in WZYD-N. Moreover, 6-dehydrogingerdione was low in both WZYD-N and WZYD-S. Preliminary pharmacodynamic experiments showed that WZYD-N could reduce the number of writhing responses and inhibit pain responses induced by acetic acid in mice, exhibiting analgesic effects similar to the WZYD group. WZYD-S could reduce the absorbance value of the intraperitoneal lavage fluid in mice, exhibiting anti-inflammatory effects comparable to the WZYD group. Network pharmacology analysis indicated that dehydroevodiamine, rutaecarpine, 6-gingerol, and ginsenoside Rg_1 might be the analgesic active components of WZYD, and limonin, rutaevine, and ginsenoside Ro might be the anti-inflammatory active components of WZYD. This study proposed a novel strategy for elucidating the pharmacodynamic basis of WZYD and innovating classical formulas.
Animals ; Analgesics/pharmacology* ; Drugs, Chinese Herbal/pharmacology* ; Mice ; Anti-Inflammatory Agents/pharmacology* ; Male ; Chromatography, High Pressure Liquid ; Tandem Mass Spectrometry ; Pain/drug therapy* ; Humans

Objectives: It is unclear whether G protein-coupled receptor 61 (GPR61) affecting body weight, plays a role in the association between birth weight and weather. This study aimed to assess the effects of prenatal weather and GPR61 on birth weight. Methods: A total of 567 mother-newborn pairs were recruited in Houzhai Center Hospital during 2011-2012. We detected the maternal and neonatal GPR61 promoter methylation levels, and obtained meteorological and air pollution data. Results: A positive association was observed between maternal and neonatal GPR61 methylation levels, and both of them were affected by precipitation, relative humidity (RH) and daily temperature range (DTR). Birth weight was associated negatively with RH and positively with DTR ( P < 0.05). A significant association was observed between birth weight and neonatal GPR61 methylation. We observed that maternal GPR61 methylation seemed to modify associations between weather and birth weight ( P _interaction < 0.10), while neonatal GPR61 methylation mediated the effects of RH and DTR on birth weight ( P < 0.05). Conclusions Our findings revealed the significant associations among prenatal weather, GPR61 methylation and birth weight. Maternal GPR61 methylation may modify the susceptibility of birth weight to prenatal weather conditions, while neonatal GPR61 methylation may be a bridge of the effects of prenatal RH and DTR on birth weight.
Air Pollution/analysis* ; Birth Weight ; Female ; Humans ; Infant, Newborn ; Nerve Tissue Proteins ; Pregnancy ; Receptors, G-Protein-Coupled/metabolism* ; Temperature ; Weather

OBJECTIVE: To investigate whether blood-brain barrier (BBB) served a key role in the edema-relief effect of bloodletting puncture at hand twelve Jing-well points (HTWP) in traumatic brain injury (TBI) and the potential molecular signaling pathways. METHODS: Adult male Sprague-Dawley rats were assigned to the sham-operated (sham), TBI, and bloodletting puncture (bloodletting) groups (n=24 per group) using a randomized number table. The TBI model rats were induced by cortical contusion and then bloodletting puncture were performed at HTWP twice a day for 2 days. The neurological function and cerebral edema were evaluated by modified neurological severity score (mNSS), cerebral water content, magnetic resonance imaging and hematoxylin and eosin staining. Cerebral blood flow was measured by laser speckles. The protein levels of aquaporin 4 (AQP4), matrix metalloproteinases 9 (MMP9) and mitogen-activated protein kinase pathway (MAPK) signaling were detected by immunofluorescence staining and Western blot. RESULTS: Compared with TBI group, bloodletting puncture improved neurological function at 24 and 48 h, alleviated cerebral edema at 48 h, and reduced the permeability of BBB induced by TBI (all P<0.05). The AQP4 and MMP9 which would disrupt the integrity of BBB were downregulated by bloodletting puncture (P<0.05 or P<0.01). In addition, the extracellular signal-regulated kinase (ERK) and p38 signaling pathways were inhibited by bloodletting puncture (P<0.05). CONCLUSIONS Bloodletting puncture at HTWP might play a significant role in protecting BBB through regulating the expressions of MMP9 and AQP4 as well as corresponding regulatory upstream ERK and p38 signaling pathways. Therefore, bloodletting puncture at HTWP may be a promising therapeutic strategy for TBI-induced cerebral edema.

OBJECTIVE: To identify the prominent molecular signaling in acupoints and explore their roles in initiating the analgesia effect of manual acupuncture (MA). METHOD: A three-step study was conducted, the experiment 1 was a genome-wide analysis of the tissue at acupoint Zusanli (ST 36), including 12 Wistar rats which were divided into control, control+MA1, and control+MA7 groups. In the experiment 2, the paw withdrawal latency (PWL), immunohistochemistry and Western blot analysis of phospho-nuclear factor kappa B (NFκB) p65 (p-p65), phospho-NFκB p50 (p-p50) at ST 36 were performed on rats of saline, saline+MA, and complete Freund's adjuvant (CFA)+MA groups (n=6). In experiment 3, 24 rats were divided into saline+DMSO, CFA+DMSO, CFA+DMSO+MA, and CFA+BAY 11-7082+MA groups, the PWL and immunofluorescence assay of NFκB p65 at ST 36 was conducted. RESULT: (1) The gene: inhibitor of NFκB (Nfkbia), interleukin-1β (Il1b), interleukin-6 (Il6), chemokine c-x-c motif ligand 1 (Cxcl1), monocyte chemoattractant protein-1 (MCP-1/Ccl2) expressions in the control+MA7 group were significantly increased (P<0.05 or P<0.01), and the expression of NFκB p65 (Rela), NFκB p50 (Nfkb1) were increased in the control+MA7 group (P<0.05). (2) CFA+MA groups showed increased PWL from day 1 to 7 (P<0.01 vs. CFA), and the Western blot results were consistent with immunohistochemistry, the expression of NFκB p-p65 and NFκB p-p50 were significantly increased in the MA-related groups compared with control and CFA groups (P<0.05). (3) Compared with the CFA+DMSO+MA group, the PWL of the CFA+ BAY 11-7082+MA group decreased significantly and continued until day 5 and 7 (P<0.05 and P<0.01, respectively), and the NFκB p65 expression of CFA+BAY 11-7082+MA was significantly reduced compared with CFA+DMSO+MA (P<0.01). CONCLUSION Local NFκB signaling cascade in acupoint caused by MA is an important step in initiating the analgesic effect, which would provide new evidence for the initiation of MA-effect and improve the understanding of the scientific basis of acupuncture analgesia.
Acupuncture Analgesia ; Acupuncture Points ; Animals ; Electroacupuncture ; NF-kappa B/metabolism* ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; Signal Transduction

OBJECTIVE: To investigate the effects of Radix Angelicae Sinensis (RASI) and hydrocortisone combination on the murine asthma model and the mechanism. METHODS: BALB/c mice were randomly divided into control group, blood stasis model group, asthma model group, HSS group, RASI group and RASI+HSS group (=12). Ovalbumin (OVA) was used to replicate mice asthma model and hydrocortisone sodium succinate (HSS) to copy blood stasis model. Effects of RASI, HSS and their combination on hemorheology, anti-asthma (asthmatic behaviors, lung function, lung index and water content in lung tissue) were observed. and anti-asthma mechanisms The expression of relative cytokines, high-mobility group box 1 (HMGB1), toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) was detected by ELISA and immunohistochemistry respectively. RESULTS: Eight g/kg RASI, 0.05 g/kg HSS and their combination could significantly relieve asthma behavioral indicators, improve lung function, reduce lung index and water content in lung tissue, decrease the levels of TNF-α, IL-1β and IL-6 in broncho-alveolar lavage fluid (BALF), and inhibit the high expression of HMGB1, TLR4 and NF-κB in lung tissue. The improvement of lung function and the decrease in level of relative cytokines (TNF-α、IL-1βIL-6) were better in RASI+HSS group than those in RASI group and HSS group, and the inhibition of protein expression of TLR4 and NF-κB was also too. Combined administration of RASI and hydrocortisone could decrease serum thromboxane B2 (TXB2) content and blood viscosity, which were increased induced by hydrocortisone. CONCLUSIONS Combined administration of RASI and hydrocortisone have obvious anti-asthma effects and one of the mechanisms is to inhibit protein synthetization of HMGB1, TLR4 and NF-κB.The combined administration of RASI and hydrocortisone has stronger improvement of lung function than that of RASI and hydrocortisone alone, and it may be related to the inhibition of TLR4 and NF-κB synthetization. The combined administration of RASI can alleviate abnormal changes of hemorheology induced by hydrocortisone in treatment of asthma.
Animals ; Anti-Asthmatic Agents ; Asthma ; Bronchoalveolar Lavage Fluid ; Cytokines ; Hydrocortisone ; Lung ; Mice ; Mice, Inbred BALB C ; NF-kappa B

OBJECTIVE The present study was aimed to investigate the role of Wnt/β-catenin sig-naling in spinal VGLUT2 regulation and neuropathic pain. METHODS To elucidate the association be-tween VGLUT2 and neuropathic pain,we determined the expression and distribution characteristics of VGLUT2 in mice subjected to spared nerve injury(SNI),and then observed the effects of two VGLUT2 targeting shRNAs on mechanical allodynia and glutamate release.The effects of Wnt/β-catenin signal-ing on VGLUT2 expression and pain behavior were investigated by using Wnt agonist,Wnt1,and Wnt/β-catenin pathway inhibitor XAV939 in SNI mice.RESULTS SNI surgery induced significant up-regula-tion of VGLUT2 on postoperative days 7,14,and 21.Double immunofluorescence labeling of VGLUT2 with NeuN,MAP2,Iba-1,or GFAP showed that VGLUT2 was mainly expressed in neurons in the dor-sal horn of the spinal cord after SNI(NeuN,MAP2).Intrathecal administration of VGLUT2 shRNAs be-fore or after SNI surgery significantly decreased mechanical allodynia and glutamate release. Mean-while,Wnt1/β-catenin signaling increased significantly after SNI surgery.Over-expression of β-catenin in PC12 cells increased VGLUT2 protein level,intrathecal administration of Wnt agonist or Wnt1 signifi-cantly increased VGLUT2 protein expression in spinal cord, while Wnt/β-catenin pathway inhibitor XAV939 decreased VGLUT2 expression in PC12 cells and spinal cord.Additionally,intrathecal admin-istration of XAV939 7 days after SNI significantly attenuated mechanical allodynia in mice, which was in accordance with down-regulation of VGLUT2 protein levels.VGLUT2 shRNAs significantly attenuat-ed Wnt agonist or Wnt1 induced mechanical allodynia. CONCLUSION Wnt1/β-catenin signaling path-way up-regu-lates the spinal VGLUT2 expression,and this regulation is involved in neuropathic pain behavior.