Clinicopathologic features, tumor immune microenvironment and genomic landscape of EBV-related and EBV-unrelated poorly differentiated nonkeratinizing squamous cell carcinoma of the thymus.

OBJECTIVES: Knowledge regarding thymic EBV-related poorly differentiated nonkeratinizing squamous cell carcinoma (PDNKSCC), also known as lymphoepithelial carcinoma (LEC), is extremely limited due to its rarity. MATERIALS AND METHODS: This multi-institutional study enrolled 85 patients with thymic PDNKSCC. DNA in situ hybridization was performed to evaluate the EBV status of all 85 cases. Immunohistochemistry and next generation sequencing were performed to compare the differences in the clinicopathological and molecular features between EBV-related and EBV-unrelated PDNKSCC. Tumor-infiltrating lymphocytes (TILs) were also analyzed by these methods. RESULTS: The 85 cases were classified into 27 EBV-related PDNKSCCs (31.8 %) and 58 EBV-unrelated PDNKSCCs (68.2 %) according to the EBV status, and 35 Lymphoepithelioma pattern (LP) (41.2 %) and 50 desmoplastic pattern (DP) (58.8 %) according to the histological characteristics. Compared to the EBV-unrelated PDNKSCC, EBV-related PDNKSCC showed a younger patient predominance and more commonly displayed a LP subtype. Additionally, LP-type cases were divided into two groups: Group 1 (EBV-related, 20/85) and Group 2 (EBV-unrelated, 15/85); the DP-type cases were divided into Group 3 (EBV-unrelated, 43/85) and Group 4 (EBV-related, 7/85). The four Groups showed a significant association with patients' OS and PFS. EBV-related PDNKSCC had significantly higher PD-L1 + tumor cells (TCs) and PD-L1 + and CD8 + immune cells (ICs) than EBV-unrelated PDNKSCC. The tumor microenvironment immune type (TMIT) I (PDL1-Tumor+/CD8-High) was more common in EBV-related PDNKSCC, especially in Group 1(LP and EBV related) with more than 90 % cases belonged to TMIT I. Molecular analysis demonstrated that EBV-related PDNKSCC had a significantly higher tumour mutational burden and frequency of somatic mutations than EBV-unrelated cases. CONCLUSIONS: EBV-related PDNKSCC, especially the Group 1, could be a candidate for immunotherapy and EBV positivity may provide an indication for the selection of targeted therapy due to their high tumour mutational burden.

Toxicological characteristics of drinking water in two large-scale municipal water supply systems of a metropolitan city in Central China.

Drinking water safety is threatened by numerous toxic organic pollutants difficult to chemically monitor. This study aimed to determine the toxicological profiles of organic extracts (OEs) of water samples from source to tap in two drinking water supply systems in a metropolitan city, Central China, during different hydrological periods. Mortality, DNA damage, growth, and development of Caenorhabditis elegans were evaluated following exposure to OEs. The median lethal doses of OEs of drinking water samples (n = 48) ranged from 266 REF (relative enrichment factor) to > 1563 REF. When tested at a dose of 100 REF, 56.25% (27/48) of OEs induced genotoxicity, 4.17% (2/48) inhibited the growth, and 45.83% (22/48) decreased the offspring number in C. elegans. No clear temporal-spatial variation patterns of the OEs toxicity indicators were observed. The correlations among the toxicity indicators were generally poor. The observed toxicities were not closely related to the level of dissolved organic carbon in drinking water. These findings support using multiple endpoint bioassays, such as C. elegans-based approaches, as complementary tools to conventional chemical analysis for drinking water quality monitoring.

[Complete chloroplast genome sequencing and phylogeny of wild Atractylodes lancea from Yuexi, Anhui province].

This study investigated the choroplast genome sequence of wild Atractylodes lancea from Yuexi in Anhui province by high-throughput sequencing, followed by characterization of the genome structure, which laid a foundation for the species identification, analysis of genetic diversity, and resource conservation of A. lancea. To be specific, the total genomic DNA was extracted from the leaves of A. lancea with the improved CTAB method. The chloroplast genome of A. lancea was sequenced by the high-throughput sequencing technology, followed by assembling by metaSPAdes and annotation by CPGAVAS2. Bioiformatics methods were employed for the analysis of simple sequence repeats(SSRs), inverted repeat(IR) border, codon bias, and phylogeny. The results showed that the whole chloroplast genome of A. lancea was 153 178 bp, with an 84 226 bp large single copy(LSC) and a 18 658 bp small single copy(SSC) separated by a pair of IRs(25 147 bp). The genome had the GC content of 37.7% and 124 genes: 87 protein-coding genes, 8 rRNA genes, and 29 tRNA genes. It had 26 287 codons and encoded 20 amino acids. Phylogenetic analysis showed that Atractylodes species clustered into one clade and that A. lancea had close genetic relationship with A. koreana. This study established a method for sequencing the chloroplast genome of A. lancea and enriched the genetic resources of Compositae. The findings are expected to lay a foundation for species identification, analysis of genetic diversity, and resource conservation of A. lancea.

Electroacupuncture relieves hyperalgesia by regulating neuronal-glial interaction and glutamate transporters of spinal dorsal horns in rats with acute incisional neck pain.

Objective: Glial cells are involved in the analgesic effect of electroacupuncture (EA) in rats with chronic neurological pain. The objective of this study was to observe the role of neuronal-glial interaction and glutamate (Glu) transporters in EA-induced acute neck pain relief in rats. Materials and methods: Male rats were placed into the following five groups: control, model, EA Futu (LI18), EA Hegu (LI4)-Neiguan (PC6), and EA Zusanli (ST36)-Yanglingquan (GB34). The incisional neck pain model was established by making a longitudinal incision along the midline of the neck. The thermal pain threshold (TPT) was measured using a radiation heat detector. The immunoactivities of glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule 1 (Iba-1), neurokinin-1 receptor (NK-1R), Glu aspartate transporter (GLAST), and Glu transporter-1 (GLT-1) in the dorsal horns (DHs) of the cervico-spinal cord (C2-C5) were detected using immunofluorescence histochemistry. The expression levels of GFAP, Iba-1, GLAST, and GLT-1 mRNAs were determined using quantitative real-time polymerase chain reaction (PCR). Results: The TPT and levels of mRNAs expression and immunoactivity of GLT-1 and GLAST were significantly decreased, and those of Iba-1 and GFAP were significantly increased in the model group than those of the control group (P < 0.05). The activated microgliacytes were gathered around the NK-1R positive neurons, and co-expression of NK-1R and astrocytes was observed in the model group. EA LI18 significantly increased the TPT and expression of GLAST and GLT-1 mRNAs (P < 0.05) and notably decreased the number of Iba-1 positive cells and Iba-l mRNA expression (P < 0.05), whereas GLAST and GLT-1 antagonists inhibited the analgesic effect of EA LI18. However, these effects, except for the downregulation of Iba-1 mRNA, were not observed in the EA ST36-GB34 group. Fewer NK-1R-positive neurons were visible in the spinal DHs in the EA LI18 group, and the co-expression of NK-1R and astrocytes was also lower than that in the three EA groups. Conclusion: Electroacupuncture of LI18 had an analgesic effect in rats with neck incisions, which may be related to its functions in suppressing the neuronal-glial cell interaction through NK-1R and upregulating the expression of GLAST and GLT-1 in the spinal DHs.

The role of PTEN in primary sensory neurons in processing itch and thermal information in mice.

PTEN is known as a tumor suppressor and plays essential roles in brain development. Here, we report that PTEN in primary sensory neurons is involved in processing itch and thermal information in adult mice. Deletion of PTEN in the dorsal root ganglia (DRG) is achieved in adult Drg11-CreER: PTENflox/flox (PTEN CKO) mice with oral administration of tamoxifen, and CKO mice develop pathological itch and elevated itch responses on exposure to various pruritogens. PTEN deletion leads to ectopic expression of TRPV1 and MrgprA3 in IB4+ non-peptidergic DRG neurons, and the TRPV1 is responsive to capsaicin. Importantly, the elevated itch responses are no longer present in Drg11-CreER: PTENflox/flox: TRPV1flox/flox (PTEN: TRPV1 dCKO) mice. In addition, thermal stimulation is enhanced in PTEN CKO mice but blunted in dCKO mice. PTEN-involved regulation of itch-related gene expression in DRG neurons provides insights for understanding molecular mechanism of itch and thermal sensation at the spinal level.

[κ-opioid receptor is involved in electroacupuncture analgesia via inhibition of spinal microglial Toll-like receptor 4 in neuropathic pain rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the expression of lumbar spinal κ-opioid receptor (KOR) and Toll-like receptor 4(TLR4) in microglia in neuropathic pain rats, so as to explore the role of cross-talk between KOR and TLK4 in EA-induced alleviation of chronic neuropathic pain. METHODS: Wistar male rats were randomized into control, model, EA and EA plus KOR inhibitor (EA+inhibitor) groups (n=18 in each group). The neuropathic pain model was established in rats by ligature of the right sciatic nerve. EA was applied at bilateral "Zusanli"(ST36) and "Yanglingquan"(GB34) for 30 min, once daily for 5 days. JDTic dihydrochloride (a KOR inhibitor) was administrated by intraperitoneal injection before EA intervention. The difference value of paw withdrawal thermal latency (PWLD) of the bilateral hind-limbs was used as the thermal pain reaction level. At the end of experiments, the rat's lumbar spinal cord (L2-L4) was taken for detecting the expression of CD68 mRNA (a marker of the activated microglia) and Iba-1 (a marker for the activated and resting microglia) immunoactivity, and dynorphin content, and KOR mRNA and TLR4 protein (in immunomagnetic microbead method separated microglia) by using fluorescence quantitative PCR, immunofluorescence, radioimmunoassay and Western blot, separately. RESULTS: Compared with the control group, a strong thermal hyperalgesia was induced, the expression levels of Iba-1 and CD68 mRNA in the spinal cord, TLR4 protein of the spinal microglia were significantly increased(P<0.01) in the model group. The microglia were characterized by somatic hypertrophy and thickened branches in the model group. After EA intervention, the PWLD, the expression of Iba-1, CD68 mRNA and TLR4 protein of the microglia were significantly decreased(P<0.05), while the content of spinal dynorphin and the expression of KOR mRNA of the microglia increased in the EA group relative to the model group(P<0.05). The hypertrophic microglia shrinked slightly in the EA group. After injection of KOR inhibitor, the PWLD and expression levels of Iba-1, CD68 mRNA and TLR4 protein were significantly increased(P<0.05), and the expression of KOR mRNA was significantly decreased(P<0.05) in the EA+inhibitor group in comparison with the EA group. CONCLUSION: The analgesia effect of EA may partly mediated by spinal microglial KOR and the activation of KOR of microglia may be a target for inhibition of microglial TLR4-induced pro-inflammatory signaling.

Total organic halogen in two drinking water supply systems: Occurrence, variations, and relationship with trihalomethanes.

The spatiotemporal presence of overall disinfection by-products (DBPs) in two full-scale drinking water supply systems (DWSSs) were investigated using quantification of total organic halogen (TOX). The relationships of TOX with water quality parameters (especially the most regulated DBPs, trihalomethanes (THMs)) were also evaluated. The TOX levels ranged between 2.6 and 70.3 µg Cl/L and between 46.6 and 205.9 µg Cl/L in raw water and distribution water, respectively. The TOX concentration in water increased by an average of nine times after water treatment and varied slightly during distribution, suggesting that TOX in drinking water was mainly formed during chlorination disinfection rather than distribution. No clear seasonality in TOX level was observed. Positive correlations were found between raw water dissolved organic carbon (DOC) with an increase in TOX in treated water and between DOC level with TOX content in distributed water, emphasizing a key role of organics in TOX formation. Chloroform (TCM) was the dominant THM, followed by bromodichloromethane (BDCM) in the drinking water, and the levels of the other two measured THMs (dibromochloromethane and bromoform) were negligible. THM2 (sum of TCM and BDCM) made up average of 18% of the TOX, and was weakly correlated with TOX content (rs = 0.321; P < 0.05), implying that THM is not a suitable surrogate measure for TOX in drinking water. This study provides basic data on the occurrence and variation of TOX within conventional DWSSs and highlights the importance of using TOX measurements to obtain more accurate information about DBP occurrence, for exposure assessment and regulatory determination.

Hepatic Vps33b deficiency aggravates cholic acid-induced cholestatic liver injury in male mice.

Vacuolar protein sorting 33B (VPS33B) is important for intracellular vesicular trafficking process and protein interactions, which is closely associated with the arthrogryposis, renal dysfunction, and cholestasis syndrome. Our previous study has shown a crucial role of Vps33b in regulating metabolisms of bile acids and lipids in hepatic Vps33b deficiency mice with normal chow, but it remains unknown whether VPS33B could contribute to cholestatic liver injury. In this study we investigated the effects of hepatic Vps33b deficiency on bile acid metabolism and liver function in intrahepatic cholestatic mice. Cholestasis was induced in Vps33b hepatic knockout and wild-type male mice by feeding 1% CA chow diet for 5 consecutive days. We showed that compared with the wild-type mice, hepatic Vps33b deficiency greatly exacerbated CA-induced cholestatic liver injury as shown in markedly increased serum ALT, AST, and ALP activities, serum levels of total bilirubin, and total bile acid, as well as severe hepatocytes necrosis and inflammatory infiltration. Target metabolomics analysis revealed that hepatic Vps33b deficiency caused abnormal profiles of bile acids in cholestasis mice, evidenced by the upregulation of conjugated bile acids in serum, liver, and bile. We further demonstrated that the metabolomics alternation was accompanied by gene expression changes in bile acid metabolizing enzymes and transporters including Cyp3a11, Ugt1a1, Ntcp, Oatp1b1, Bsep, and Mrp2. Overall, these results suggest a crucial role of hepatic Vps33b deficiency in exacerbating cholestasis and liver injury, which is associated with the altered metabolism of bile acids.

[Spinal cord Toll like receptor 4 and its co-stimulatory molecule heat shock protein 90 may parti-cipate in electroacupuncture analgesia in rats with chronic neuropathic pain].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the expression of Toll like receptor 4(TLR4)and heat shock protein 90(HSP90) in the spinal cord of rats with chronic constriction injury (CCI) of sciatic nerve, so as to explore the mechanism of spinal cord TLR4 and HSP90 in alleviating chronic neuropathic pain by EA. METHODS: Male Wistar rats were randomized into control, model, EA, HSP90 inhibitor (inhibitor) and EA+ inhibitor groups (n=10 in each group). The neuropathic pain model was established by ligature of the right sciatic nerve to induce CCI. EA (1 mA，2 Hz/15 Hz)was applied at bilateral "Zusanli"(ST36) and "Yanglingquan"(GB34) for 30 min, once daily for 5 days. Rats of the inhibitor and EA+inhibitor groups were given a subcutaneous injection of HSP90 inhibitor geldanamycin (50 µg/kg) at the neck before daily EA. The paw withdrawal latency (PWL) of the bilateral hind-limbs was detected by using an algesia-detector. The contents of interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α) in the lumbar spinal cord (L2-L4) tissue were detected by enzyme-linked immunosorbent assay. The relative expression levels of HSP90 and TLR4 proteins in the lumbar spinal cord (L2-L4) were detected using Western blot and immunofluorescence double labeling, respectively. RESULTS: Following CCI, a strong thermal hyperalgesia, an apparent up-regulation of expression of HSP90 and TLR4 proteins and TLR4 in microglia, and increasing levels of IL-1ß and TNF-α in the spinal cord were induced in the model group relevant to the control group (P<0.01，P<0.05). Five sessions of EA intervention or inhibitor injection significantly attenuated hyperalgesia, reversed the increase of IL-1ß and TNF-α, and down-regulated the expression of TLR4 in microglia (P<0.05). Compared with the model group, the expression of HSP90 was further increased (P<0.05), and those of TLR4 in microglia and neurons were significantly decreased and increased, respectively in the EA group (P<0.05). Compared with the EA group, the levels of PWLD,TLR4 and HSP90 expression, and the proportions of neuronal nuclei antigen（NeuN） and TLR4, and ionized calcium binding adapter molecule （Iba1） and TLR4 co-expressed cells were significantly decreased in the inhibitor group and EA+inhibitor group (P<0.05). The proportion of NeuN and TLR4 co-expression cells in the EA+inhibitor group was significantly higher than that of the inhibitor group (P<0.05). CONCLUSION: EA stimulation of ST36 and GB34 can alleviate thermal hyperalgesia in CCI rats, which is closely associated with its effect in regulating the expression of TLR4 in the spinal cord neurons and microglia. HSP90 in the spinal cord may be a co-stimulatory molecule for EA induced relief of neuropathic pain by regulating TLR4.

Discovery of hCES2A inhibitors from Glycyrrhiza inflata via combination of docking-based virtual screening and fluorescence-based inhibition assays.

Human carboxylesterase 2 (hCES2A) is a key target to ameliorate the intestinal toxicity triggered by irinotecan that causes severe diarrhea in 50%-80% of patients receiving this anticancer agent. Herbal medicines are frequently used for the prevention and treatment of the intestinal toxicity of irinotecan, but it is very hard to find strong hCES2A inhibitors from herbal medicines in an efficient way. Herein, an integrated strategy via combination of chemical profiling, docking-based virtual screening and fluorescence-based high-throughput inhibitor screening assays was utilized. Following the screening of a total of 73 herbal products, licorice (the dried root of Glycyrrhiza species) was found with the most potent hCES2A inhibition activity. Further investigation revealed that the chalcones and several flavonols in licorice displayed strong hCES2A inhibition activities, while isoliquiritigenin, echinatin, naringenin, gancaonin I and glycycoumarin exhibited moderate inhibition of hCES2A. Inhibition kinetic analysis demonstrated that licochalcone A, licochalcone C, licochalcone D and isolicoflavonol potently inhibited hCES2A-mediated fluorescein diacetate hydrolysis in a reversible and mixed inhibition manner, with Ki values less than 1.0 µM. Further investigations demonstrated that licochalcone C, the most potent hCES2A inhibitor identified from licorice, dose-dependently inhibited intracellular hCES2A in living HepG2 cells. In summary, this study proposed an integrated strategy to find hCES2A inhibitors from herbal medicines, and our findings suggested that the chalcones and isolicoflavonol in licorice were the key ingredients responsible for hCES2A inhibition, which would be very helpful to develop new herbal remedies or drugs for ameliorating hCES2A-associated drug toxicity.

GABAergic Inhibition of Spinal Cord Dorsal Horns Contributes to Analgesic Effect of Electroacupuncture in Incisional Neck Pain Rats.

BACKGROUND: Acupuncture has shown to be effective in relieving post-surgical pain. Nonetheless, its underlying mechanisms remain largely unknown. In the present study, we investigated the effect of electroacupuncture (EA) on the expression of GABA, GABA-A receptor (R) and GABA-BR in the spinal cord dorsal horns (DHs), and the involved neural cells in rats with incisional neck pain. MATERIALS AND METHODS: Male SD rats were randomly divided into control, model, Futu (LI18), Hegu-Neiguan (LI4-PC6), and Zusanli-Yanglingquan (ST36-GB34) groups. The incisional neck pain model was established by making a longitudinal incision and repeated mechanical separation along the thyroid gland region. EA (2Hz/100Hz, 1mA) was applied to LI18, LI4-PC6, ST36-GB34 separately for 30min, once at 4, 24 and 48h after incision. The local thermal pain threshold (TPT) of the focus was measured and the expression of GABA, and GABAR proteins and mRNAs detected by immunofluorescence stain and quantitative RT-PCR, respectively. RESULTS: The analgesic effect of LI18 and LI4-PC6 was superior to that of ST36-GB34 in incisional neck pain rats. Moreover, the EA stimulation of LI18 or LI4-PC6 increased the expression of GABA and GABA-Aα2 and GABA-Aß3, GABA-B1, and GABA-B2 mRNAs in spinal DHs 4h after surgery, while GABA-A and GABA-B antagonists inhibited the analgesic effect of LI18. Immunofluorescence double staining showed that GABA was expressed on astrocytes and neurons, and GABA-B expressed only on neurons. CONCLUSION: EA of both LI18 and LI4-PC6 has a good analgesic effect in incisional neck pain rats, which is closely related to their effects in upregulating the expression of GABA and its receptors in spinal DHs. The effects of LI18 and LI4-PC6 EA are obviously better that those of ST36-GB34 EA, and GABA is expressed on neurons and astrocytes.

Inhibition of pancreatic lipase by environmental xenoestrogens.

Environmental xenoestrogens are the most accessible endocrine disrupting chemicals that have been reported with harmful effects on human health. Although the influences of xenoestrogens on the endocrine system have been extensively studied, it remains unclear whether these xenoestrogens can affect the digestive system in mammals. This study aimed to investigate the inhibitory effects and the underlying mechanism of six non-steroidal synthetic estrogens (including hexestrol, diethylstilbestrol, dienestrol, bisphenol A, bisphenol AF and bisphenol Z) on pancreatic lipase (PL), a key digestive enzyme responsible for lipid digestion and absorption in mammals. The results clearly demonstrated that hexestrol, diethylstilbestrol and dienestrol exhibited strong inhibition on PL, with the IC50 values of less than 1.0 µM. Further investigations elucidated that these three synthetic estrogens functioned as mixed inhibitors of PL, with the Ki values of less than 1 µM. Moreover, molecular dynamics simulations showed that diethylstilbestrol and its analogues might block the binding of substrate on PL via occupying the portal to the active site of PL and thereby inhibit the hydrolytic activity of this key enzyme. Collectively, these results suggested that diethylstilbestrol and its analogues were potent PL inhibitors, which might play a profound role in lipid absorption and weight gain in mammals.

An investigation of changes in water quality throughout the drinking water production/distribution chain using toxicological and fluorescence analyses.

Changes in water quality from source water to finished water and tap water at two conventional drinking water treatment plants (DWTPs) were monitored. Beside the routine water quality testing, Caenorhabditis elegans-based toxicity assays and the fluorescence excitation-emission matrices technique were also applied. Both DWTPs supplied drinking water that met government standards. Under current test conditions, both the investigated finished water and tap water samples exhibited stronger lethal, genotoxic and reprotoxic potential than the relative source water sample, and the tap water sample was more lethal but tended to be less genotoxic than the corresponding finished water sample. Meanwhile, the nearly complete removal of tryptophan-like substances and newly generated tyrosine-like substances were observed after the treatment of drinking water, and humic-like substances were identified in the tap water. Based on these findings, toxic pollutants, including genotoxic/reproductive toxicants, are produced in the drinking water treatment and/or distribution processes. Moreover, further studies are needed to clarify the potentially important roles of tyrosine-like and humic-like substances in mediating drinking water toxicity and to identify the potential sources of these contaminants. Additionally, tryptophan-like fluorescence may be adopted as a useful parameter to monitor the treatment performance of DWTPs. Our observations provided insights into the importance of utilizing biotoxicity assays and fluorescence spectroscopy as tools to complement the routine evaluation of drinking water.

[Comparing the effects of fast and slow expansion on nasal cavity and maxilla structure].

OBJECTIVE: This study aims to compare the effects of fast and slow expansion on nasal cavity structure. METHODS: A total of 40 patients were selected and randomly divided into two groups. Cone-beam computer tomography (CBCT) was obtained before and after surgery and used for comparing the changes in nasal structure before and after treatment. RESULTS: Fast expansion had resulted in greater changes in the basilar and nasal bone arch extension structures than slow expansion. No significant difference at maxillary width and nasal parenchyma. CONCLUSIONS: Rapid expansion therapy has more beneficial effects on nasal function.

[Electroacupuncture alleviated incisional neck pain possibly by suppressing TNF-α expression and increasing IL-4/IL-4 receptor signaling in cervical dorsal part of spinal cord in incisio-nal neck pain rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on incisional pain and expression of tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10) and interleukin-4 (IL-4) of cervical dorsal part of spinal cord in rats with incisional neck pain, so as to explore its analgesic mechanisms. METHODS: Eighty-four male SD rats were randomly divided into normal control, model, EA-Futu(LI18) and EA-Zusanli(ST36)-Yanglingquan(GB34, EA-ST36-GB34) groups (n=21 in each group). The incisional neck pain model was established by making a longitudinal incision along the bilateral cervical thyroid regions and repeated mechanical separation stimulation. For rats of the EA groups, EA (2 Hz/100 Hz, 1 mA) was applied to bilateral LI18 or ST36-GB34 for 30 min/ time during the surgery, and 20 and 44 h after surgery, respectively. The thermal pain threshold (TPT) of the incisional region was detected. The immunoactivity of TNF-α and IL-10 of the dorsal portion of the cervical spinal cord (C2-C5) was detected by immunofluorescence, and the expression of TNF-α, IL-10, IL-4 and IL-4 receptor (IL-4R) mRNAs was determined by quantitative real-time PCR. RESULTS: Compared with the normal group, the TPT of the incisional area was significantly decreased at 4, 24 and 48 h after neck-incision (P<0.05), the levels of TNF-α mRNA, IL-10 mRNA and TNF-α IL-10 immunoactivity at 24 h were remarkably increased (P<0.05), and the expression of IL-4R mRNA was considerably decreased at 24 h in the model group (P<0.05). Following EA intervention, the TPT, and expression levels of IL-4 mRNA and IL-4R mRNA were significantly increased at 24 h after surgery in the EA-LI18 group relevant to the model group (P<0.05), while the expression level of TNF-α(coexpressed with microgliacytes) in the EA-LI18 group, and TNF-α mRNA expression at 24 h in both EA-LI18 and EA-ST36-GB34 groups, as well as the expression of IL-10 and IL-10 mRNA at 24 h in both EA-LI18 and EA-ST36-GB34 groups were significantly decreased (P<0.05). The effect of EA LI18 was significantly superior to that of EA ST36-GB34 in up-regulating TPT and expression of IL-4 mRNA and IL-4R mRNA at 24 h (P<0.05). CONCLUSION: EA of LI18 has an analgesic effect in incisional neck pain rats, which may be related to its effect in down-regulating the expression of TNF-α, IL-10 and promoting IL-4 /IL-4R signaling in dorsal portions of the cervical spinal cord. The analgesic effect of EA LI18 is better than that of EA ST36-GB34.

[Expression of MiR-155 in Tissue of Patients with Diffuse Large B-Cell Lymphoma and Its Effect on Cell Biological Characteristics].

OBJECTIVE: To investigate the expression of miR-155 in patients with diffuse large B-cell lymphoma (DLBCL), and to explore the effect of transfection of miR-155 inhibitor on the biological characteristics of DLBCL cells. METHODS: A total of 76 patients with DLBCL treated in our hospital were selected from April 2013 to December 2017. In the same time, 40 cases of lymph node reactive hyperplasia (LNRH) were selected as control group. DB cells were cultured and divided into miR-155 inhibitor, negative control and blank groups. The expressions of miR-155 in DLBCL, negative and blank control groups were detected by using real-time PCR, the cell proliferation was detected by MTT assay, the apoptosis was detected by flow cytometry, and the cell migration and invasion were detected by Transwell assay. RESULTS: The relative expression level of miR-155 in tissues of DLBCL patients was significantly higher than that in tissne of controls (1.93±0.16 vs 1.01±0.09) (t=33.991, P=0.000). The expression level of miR-155 increased (P＜0.05) in DLBCL patients with LDH level abnormarity, BCL-2+, MUM1+, Ki-67≥50%, non-GC type, Ann Arbor stage III-IV, extranodal lesion number≥2 and IPI score 3-5. The relative expression level of miR-155 in the miR-155 inhibitor group was lower than that in the negative control group and the blank group (P＜0.05). The absorbance (A) values at 24, 48, 72 and 96 h of culture in the miR-155 inhibitor group were lower than those in the negative control group and the blank group (P＜0.05), while the apoptotic rate was higher than that in the negative control group and the blank group (P＜0.05). Both the migrating cells and invading cell number in the miR-155 inhibitor group were lower than those in the negative control group and the blank group (P＜0.05). CONCLUSION: The miR-155 highly expresses in DLBCL tissue, which relates with tumor malignancy and invasion progression. The specific inhibition of miR-155 expression in DB cells can reduce cell proliferation, accelerate cell apoptosis, and inhibit cell migration and invasion.

Contribution of GABAergic modulation in DRGs to electroacupuncture analgesia in incisional neck pain rats.

PURPOSE: Acupuncture therapy is effective for relieving postoperative pain. Our previous study showed that electroacupuncture (EA) at Futu (LI18) and Hegu (LI4)-Neiguan (PC6) could alleviate incisional neck pain, which was related with its effect in upregulating γ-aminobutyric acid (GABA) expression in cervical (C3-6) dorsal root ganglions (DRGs); but whether its receptor subsets GABAAα2R and GABABR1 in C3-6 DRGs are involved in EA analgesia or not, it remains unknown. MATERIALS AND METHODS: Seventy-five male Sprague Dawley rats were randomized to normal control, model, LI18, LI4-PC6, and Zusanli (ST36)-Yanglingquan (GB34) groups. The incisional neck pain model was established by making a longitudinal incision along the midline of the rats' neck, followed by repeated mechanical stimulation. EA was applied to bilateral LI18, LI4-PC6, or ST36-GB34 for 30 minutes at 4, 24, and 48 hours after operation. The thermal pain threshold of the neck was detected by a tail-flick unit, and the C3-6 DRGs were removed for assaying the immunoactivity of substance P (SP), GABAAα2R, glial fibrillary acidic protein (GFAP; a marker of satellite glial cells [SGCs]), and GABABR1 and the expression of GABAAα2R and GABABR1 mRNA and proteins using immunofluorescence, real-time PCR, and Western blotting, respectively. RESULTS: The cervical thermal pain threshold was significantly lower in the model group than the normal group (P<0.001), indicating hyperalgesia after neck incision, and was considerably increased in both EA-LI18 and LI4-PC6 groups (P<0.001), but not in ST36-GB34 group compared with model group (P>0.05). Immunofluorescence staining showed that GABAAα2 R expressed on SP+ neurons, and GABABR1 on SGCs. EA of LI18 and LI4-PC6 markedly suppressed the modeling-induced upregulation of the immunoactivity of SP (P<0.001 and P<0.01, respectively) and GFAP (P<0.01 and P<0.001, respectively) and significantly reversed neck incision-induced downregulation of the expression of GABAAα2R and GABABR1 mRNAs and proteins (P<0.05). CONCLUSION: EA of LI18 and LI4-PC6 has an analgesic effect in incisional neck pain rats, which is related to its effects in upregulating GABAergic inhibitory modulation on nociceptive peptidergic neurons and SGCs in cervical DRGs.

[High Mobility Group Box 1/ CD 24 Receptor/ß-EP Signaling in "Zusanli" (ST 36) Region Contributes to Electroacupuncture Analgesia in Rats with Neuropathic Pain].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the expression of high mobility group box 1 (HMGB 1) and its receptor CD 24 proteins and ß-endorphin (ß-EP) content in "Zusanli" (ST 36) region in rats with chronic constriction injury (CCI), so as to explore its mechanisms underlying pain relief. METHODS: Thirty male Wistar rats were rando-mized into control, CCI model and EA groups (n＝ 10 rats in each). The neuropathic pain model was established by ligature of the left sciatic nerve to induce CCI in the model and EA groups, and sham operation was performed in rats of the control group. Paw with drawal latency (PWL, thermal pain threshold) of the bilateral hind-limbs was detected by using an algesia-detector. Eight days after CCI operation, EA was applied to bilateral "Zusanli" (ST 36) and "Yanglingquan" (GB 34) for 30 min, once daily for 5 days. The acetylated-HMGB 1 expression was determined by immunoprecipitation, and the expression of HMGB 1 and toll like receptor 4 (TLR 4) proteins and CD 24 mRNA were detected using Western blot and fluorescent quantitative real time-PCR, respectively, and the content of ß-EP in the acupoint region was assayed by enzyme linked immunosorbent assay (ELISA). Anti-CD 24 neutralizing antibody (200 µL, 100 µg/mL) was injected into ST 36 region once daily for 3 days for verifying the involvement of HMGB 1/CD 24 signaling in EA analgesia. RESULTS: Compared with the control group, the bilateral PWL difference values in the other two groups were significantly increased (P<0.05), meaning an occurrence of hyperalgesia after CCI. In comparison with the CCI model group, the hyperalgesia in the EA group was obviously decreased (P<0.05). After CCI, the expression levels of HMGB 1 and TLR 4 proteins were considerably increased compared with those in the control group (P<0.05). After 5-times' EA, the acetylated-HMGB 1, the expression of CD 24 mRNA, and the content of ß-EP were notably up-regulated (P<0.05), and there were no obvious changes in the expression levels of HMGB 1 and TLR 4 proteins (P>0.05). After local injection of anti-CD 24 antibody, EA-induced increases of ß-EP content and reduction of thermal pain threshold were significantly suppressed (P<0.05). CONCLUSION: EA of ST 36 and GB 34 can alleviate neuropathic pain in CCI rats, which is associated with its effects in up-regulating ß-EP content, and HMGB 1 protein and CD 24 mRNA expression levels in ST 36 region. The activated HMGB 1/CD 24/ß-EP signaling contributes to EA-ST 36 induced analgesia.