Electroacupuncture activated local sympathetic noradrenergic signaling to relieve synovitis and referred pain behaviors in knee osteoarthritis rats.

Introduction: Recent research has focused on the local control of articular inflammation through neuronal stimulation to avoid the systemic side effects of conventional pharmacological therapies. Electroacupuncture (EA) has been proven to be useful for inflammation suppressing and pain reduction in knee osteoarthritis (KOA) patients, yet its mechanism remains unclear. Methods: In the present study, the KOA model was established using the intra-articular injection of sodium monoiodoacetate (MIA) (1 mg/50 µL) into the knee cavity. EA was delivered at the ipsilateral ST36-GB34 acupoints. Hind paw weight-bearing and withdrawl thresholds were measured. On day 9, the histology, dep enrichment proteins, cytokines contents, immune cell population of the synovial membrane of the affected limbs were measured using HE staining, Masson staining, DIA quantitative proteomic analysis, flow cytometry, immunofluorescence staining, ELISA, and Western Blot. The ultrastructure of the saphenous nerve of the affected limb was observed using transmission electron microscopy on the 14th day after modeling. Results: The result demonstrated that EA intervention during the midterm phase of the articular inflammation alleviated inflammatory pain behaviors and cartilage damage, but not during the early phase. Mid-term EA suppressed the levels of proinflammatory cytokines TNF-α, IL-1ß, and IL-6 in the synovium on day 9 after MIA by elevating the level of sympathetic neurotransmitters Norepinephrine (NE) in the synovium but not systemic NE or systemic adrenaline. Selective blocking of the sympathetic function (6-OHDA) and ß2-adrenergic receptor (ICI 118,551) prevented the anti-inflammatory effects of EA. EA-induced increment of the NE in the synovium inhibited the CXCL1-CXCR2 dependent overexpression of IL-6 in the synovial macrophages in a ß2-adrenergic receptor (AR)-mediated manner. Discussion: These results revealed that EA activated sympathetic noradrenergic signaling to control local inflammation in KOA rats and contributed to the development of novel therapeutic neurostimulation strategies for inflammatory diseases.

[Effect of electroacupuncture on spontaneous pain during the synovial inflammatory response stage and allodynia at the later stage in rats with knee osteoarthritis].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on pain behavior, synovial inflammatory response and demyelination of saphenous nerve in the rats modeled with knee osteoarthritis (KOA) and explore the effect mechanism of EA for reliving allodynia. METHODS: Eighty-four male SD rats were randomly divided into a control group, a model group and an EA group, 28 rats in each one. Intra-articular injection of sodium monoiodoacetate (MIA) was administered in right knee joint of each rat in the model group and the EA group to establish the KOA model. In the EA group, separately, on day 5, 7 and 9 after modeling, EA was applied at "Zusanli" (ST 36) and "Yanglingquan" (GB 34) on the right side, with disperse-dense wave (2 Hz/15 Hz), 1 mA in current intensity, for 30 min in one intervention, once a day, and 3 interventions were required. On the 9th day after modeling, the weight-bearing rate was calculated for the affected limbs of the rats in each group, the synovial morphological changes were observed using HE and Masson staining, flow cytometry was adopted to detect the synovial immunocyte counts, and MSD multi-spot assay was used to detected the synovial inflammatory cytokine content. On the 14th day after modeling, the hind-paw mechanical withdrawal threshold was observed in each group and the ultrastructure of the saphenous nerve was observed under transmission electron microscopy. RESULTS: On the 9th day after modeling, compared with the control group, the weight-bearing rate of the affected limb was reduced (P<0.01), the synovial hyperplasia, inflammatory cell infiltration and synovial fibrosis occurred in the affected limb; the counts of synovial CD11b+ cells and M1 macrophages (CD11b+CD86+) were increased (P<0.01), the contents of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1ß, IL-10 and IL-13 in the synovial tissue were elevated (P<0.01, P<0.05) for the rats of the model group. Compared with the model group, the weight-bearing rate of the affected limb was increased (P<0.05), the synovial hyperplasia, inflammatory cell infiltration and synovial fibrosis were mitigated, the counts of CD11b+ cells and M1 macrophages (CD11b+CD86+) in the synovial tissue, and the contents of TNF-α and IL-6 were reduced (P<0.01, P<0.05) in the EA group. On the 14th day after modeling, the hind-paw mechanical withdrawal threshold was reduced in the model group when compared with the control group (P<0.01), and it was increased in the EA group when compared with the model group (P<0.05). Besides, in the model group, obviously, the myelin sheath structure was destroyed, the myelin layer was disintegrated and loosened, the axon was extruded or the layer thicken and cracked. Compared with the model group, the injury of saphenous nerve was alleviated remarkably in the EA group. CONCLUSION: The intervention with EA may attenuate the synovial inflammatory response and the injury of saphenous nerve in the affected limb of the rat with KOA, so that the spontaneous pain during the synovial inflammatory response stage and allodynia at the later stage are relieved.

[Sensory and sympathetic nerves are involved in the changes of skin temperature, blood infusion and inflammatory cytokines of cutaneous tissue in the sensitized area of colitis rats].

OBJECTIVE: To investigate the changes of skin temperature, blood infusion and inflammatory cytokines of cutaneous tissue in the sensitized area of colitis model rats, as well as the relationship between sensory and sympathetic nerves and the formation of sensitized area, and to initially reveal the partial physical-chemical characteristics of the sensitized area in the colitis model rats. METHODS: Thirty-five male SD rats were randomly divided into a control group (n=10), a model group (n=18) and a guanethidine group (n=7). 5% dextran sulfate sodium (DSS) was adopted for 6-day free drinking to establish colitis model in the model group and the guanethidine group. On day 6 and 7, in the guanethidine group, guanethidine solution (30 mg/kg) was injected intraperitoneally for sympathetic block. On day 7, after injection of evans blue (EB) solution, the EB extravasation areas on the body surface were observed to investigate the distribution and physical-chemical characteristics of the sensitized area. The control area was set up, 0.5 cm away from the sensitized area, and with the same nerve segment innervation. Disease activity index (DAI) score of rats was compared between the normal group and the model group, and the morphological changes in the colon tissue were investigated with HE method. Using infrared thermal imaging technology and laser speckle flow imaging technology, skin temperature and blood infusion were determined in the sensitized area and the control area of the rats in the model group. Immunofluorescence technique was adopted to observe the expression levels of the positive nerve fibers of substance P (SP), calcitonin gene-related peptide (CGRP) and tyrosine hydroxylase (TH), and the correlation with blood vessels; as well as the expression levels of SP positive nerve fibers/tryptase+ mast cells, and tryptase+ mast cells/5-hydroxytryptamine (5-HT) in skin tissue in the sensitized area and the control area of the rats in the model group. MSD multi-level factorial method and ELISA were applied to determine the contents of pro-inflammatory and anti-inflammatory cytokines (e.g. TNF-α, IL-1ß, IL-6, IL-4 and IL-10) and anti-inflammatory substance corticosterone (CORT). RESULTS: Sensitization occurred at the T12-S1 segments of the colitis model rats, especially at L2-L5 segments. Compared with the normal group, DAI score was increased in the rats of the model group (P<0.05), and the colonic mucosal damage was obvious, with the epithelial cells disordered, even disappeared, crypt destructed, submucosal edema and a large number of inflammatory cells infiltrated. In comparison with the control area, the skin temperature and blood infusion were increased in the sensitized area of the model group (P<0.05, P<0.01); as well as the expression levels of the positive nerve fibers of SP, CGRP and TH of skin tissue (P<0.05), which was specially distributed in peripheral vessels, the expression levels of SP positive nerve fibers/tryptase+ mast cells, and tryptase+ mast cells/5-HT of the skin tissue were all expanded (P<0.05) in the sensitized area of the model group. Compared with the model group, the number of sensitized areas was reduced in the guanethidine group (P<0.05). In comparison with the control area of the model group, in the sensitized area, the contents of pro-inflammatory cytokines, e.g. TNF-α, IL-1ß and IL-6, and the anti-inflammatory substance CORT of skin tissue were all increased (P<0.05); and the contents of IL-6 and TNF-α were negatively correlated with CORT (P<0.05). CONCLUSION: The sensitized areas on the body surface of colitis rats are mainly distributed in the L2-L5 segments. Sensory and sympathetic nerves are involved in the acupoint sensitization, and the sensitized areas may have the dynamic changes in pro-inflammatory and anti-inflammatory substances.

The Zur of Xanthomonas campestris functions as a repressor and an activator of putative zinc homeostasis genes via recognizing two distinct sequences within its target promoters.

It has been long considered that zinc homeostasis in bacteria is maintained by export systems and uptake systems, which are separately controlled by their own regulators and the uptake systems are negatively regulated by Zur which binds to an about 30-bp AT-rich sequence known as Zur-box present in its target promoters to block the entry of RNA polymerase. Here, we demonstrated in vivo and in vitro that in addition to act as a repressor of putative Zn(2+)-uptake systems, the Zur of the bacterial phytopathogen Xanthomonas campestris pathovar campestris (Xcc) acts as an activator of a Zn(2+) efflux pump. The Xcc Zur binds to a similar Zur-box with approximately 30-bp AT-rich sequence in the promoters of the genes encoding putative Zn(2+)-uptake systems but a 59-bp GC-rich sequence with a 20-bp inverted repeat overlapping the promoter's -35 to -10 sequence of the gene encoding a Zn(2+)-export system. Mutagenesis of the inverted repeat sequence resulted in abolishment of the in vitro binding and the in vivo and in vitro activation of the export gene's promoter by Zur. These results reveal that the Xcc Zur functions as a repressor and an activator of putative zinc homeostasis genes via recognizing two distinct sequences within its target promoters.

[Polymorphism of HLA-A, -B, -C, -DRB1 and -DQB1 Allele and Haplotype Frequency at High Resolution in Guangxi Zhuang Population].

OBJECTIVE: To analyze the characteristics of allelic and haplotypic polymorphisms of human leukocyte antigens at HLA-A, -B, -C, DRB1 and DQB1 loci in Guangxi Zhuang population. METHODS: Polymerase chain reaction-sequence based typing (PCR-SBT) was used to detect. The five loci (HLA-A, -B, -C, -DRB1, -DQB1) in 350 unrelated Zhuang ethnic individual from Guangxi region. Allelic and haplotypic frequencies were calculated by using Arlequin software 3.5.2.2. Phylogeny tree were constructed by using MEGA software 6.0, and SPSS software was used for principal component analysis. RESULTS: Among the five loci in the population, only HLA-A and DRB1 loci were observed as departures from Hardy-Weinberg expectations. A total of 19 HLA-A, 42 HLA-B, 22 HLA-C, 25 HLA-DRB1 and 15 HLA-DQB1 alleles were found in 350 samples. The most highest frequent alleles were A*11: 01(28.57%), B*46: 01(14.00%), C*01: 02(18.43%), DRB1*16: 02 (15.71%）and DQB1*05: 02 (35.00%) . The most common five loci haplotype was A*33: 03-C*03: 02-B*58: 01-DRB1*03: 01-DQB1*02: 01（6.86%）. The phylogenetic tree analysis showed that Guangxi Zhuang population had a relative close genetic relationship with southern Han Chinese populations. CONCLUSION: This reaserch found that the HLA-A, B, C, DRB1 and DQB1 loci are highly polymorphic in Guangxi Zhuang population.

HLA-B*07, HLA-DRB1*07, HLA-DRB1*12, and HLA-C*03:02 Strongly Associate With BMI: Data From 1.3 Million Healthy Chinese Adults.

Strong associations between HLA alleles and infectious and autoimmune diseases are well established. Although obesity is also associated with these diseases, the relationship between HLA and obesity has not been systematically investigated in a large cohort. In the current study, we analyzed the association of HLA alleles with BMI using data from 1.3 million healthy adult donors from the Chinese Marrow Donor Program (CMDP). We found 23 HLA alleles, including 12 low-resolution and 11 high-resolution alleles, were significantly associated with BMI after correction for multiple testing. Alleles associated with high BMI were enriched in haplotypes that were common in both Chinese and European populations, whereas the alleles associated with low BMI were enriched in haplotypes common only in Asians. Alleles B*07, DRB1*07, DRB1*12, and C*03:02 provided the strongest associations with BMI (P = 6.89 × 10-10, 1.32 × 10-9, 1.52 × 10-9, and 4.45 × 10-8, respectively), where B*07 and DRB1*07 also had evidence for sex-specific effects (Pheterogeneity = 0.0067 and 0.00058, respectively). These results, which identify associations between alleles of HLA-B, DRB1, and C with BMI in Chinese young adults, implicate a novel biological connection between HLA alleles and obesity.

[Identification of A Novel HLA-B*13:92 Allele and Analysis of Its 3D Model Structure].

OBJECTIVE: To identify a novel human leukocyte antigen (HLA) allele HLA-B*13:92 and analyze 3D model of HLA molecule. METHODS: Polymerase chain reaction sequencing-based (PCR-SBT) was used in routine HLA typing, the B locus typing results of one sample was one base mismatch with B*13:01:01, B*58:01:01 at locus 189, The Group Specific Sequencing Products (GSSP) which target at B*13 and B*58 were used to confirm difference between the new allele and highest homologous allele, then the new allele was modeled by Swiss-model to its 3D structure. RESULTS: The sequencing results showed that the new allele with highest homologous allele B*13:01:01 was the difference in the second exon at position 189 C>A (codon 39 GAC>GAA), 39 Asp (D) was changed to Glu (E). The amino acid substitution at residue 39 of the HLA polypeptide was located in α-helices of antigenic peptide-biding region. CONCLUSION: This allele is a new HLA-B allele found in Chinese Guangxi Zhang population and has been designated as HLA-B*13:92 by the World Health Organization (WHO) HLA Nomenclature Committee.

High-Resolution Analyses of Human Leukocyte Antigens Allele and Haplotype Frequencies Based on 169,995 Volunteers from the China Bone Marrow Donor Registry Program.

Allogeneic hematopoietic stem cell transplantation is a widely used and effective therapy for hematopoietic malignant diseases and numerous other disorders. High-resolution human leukocyte antigen (HLA) haplotype frequency distributions not only facilitate individual donor searches but also determine the probability with which a particular patient can find HLA-matched donors in a registry. The frequencies of the HLA-A, -B, -C, -DRB1, and -DQB1 alleles and haplotypes were estimated among 169,995 Chinese volunteers using the sequencing-based typing (SBT) method. Totals of 191 HLA-A, 244 HLA-B, 146 HLA-C, 143 HLA-DRB1 and 47 HLA-DQB1 alleles were observed, which accounted for 6.98%, 7.06%, 6.46%, 9.11% and 7.91%, respectively, of the alleles in each locus in the world (IMGT 3.16 Release, Apr. 2014). Among the 100 most common haplotypes from the 169,995 individuals, nine distinct haplotypes displayed significant regionally specific distributions. Among these, three were predominant in the South China region (i.e., the 20th, 31st, and 81sthaplotypes), another three were predominant in the Southwest China region (i.e., the 68th, 79th, and 95th haplotypes), one was predominant in the South and Southwest China regions (the 18th haplotype), one was relatively common in the Northeast and North China regions (the 94th haplotype), and one was common in the Northeast, North and Northwest China (the 40th haplotype). In conclusion, this is the first to analyze high-resolution HLA diversities across the entire country of China, based on a detailed and complete data set that covered 31 provinces, autonomous regions, and municipalities. Specifically, we also evaluated the HLA matching probabilities within and between geographic regions and analyzed the regional differences in the HLA diversities in China. We believe that the data presented in this study might be useful for unrelated HLA-matched donor searches, donor registry planning, population genetic studies, and anthropogenesis studies.

[Ambiguous allele combinations in the HLA high resolution genotyping--review].

Human lymphocyte antigen (HLA) is the most complicated human dominant polymorphic genetic system. Accurate HLA genotyping is clinically important for hematopoietic stem cell (HSC) transplantation, also important for research on many human diseases. Polymerase chain reaction-sequence based typing (PCR-SBT) provides the highest resolution level and defines new alleles, so it is widely used for HLA typing. One great disadvantage of PCR-SBT method is the fact that it cannot resolve sequences of heterozygous samples in diploid genomes, leading to ambiguous typing results which make much trouble to the accurate definition of HLA genotype. This article reviewed the occurring reasons and solution method of ambiguous allele combinations in the HLA high resolution genotyping as well as the research prospect in this field.

DNA sequencing-based typing of HPA-1 to HPA-17w systems.

Although several DNA-based human platelet antigens (HPA) typing techniques, such as PCR-SSP and PCR-SSO, have been established, the typing errors and the lack of interlaboratory reproducibility are still the issues of concerns. In the present study, polymerase chain reaction primers were designed for identification of all the phenotypically different HPA-1 to HPA-17w types by sequencing-based typing (SBT) method using genomic DNA samples. No discrepancies were observed between PCR-SSP typing and SBT typing in typing a panel of HPA-typed platelet donors that included all common HPA types and the rare HPA-1b, 2b, 3b, and 6bw homozygous donors.

[Genetic polymorphism of 9 Y-STR loci with short fragment size alleles in unrelated male individuals from Zhuang ethnic group].

The aim of this study was to investigate the genetic polymorphism of Y-chromosome specific short tandem repeat (Y-STR) loci in Zhuang ethnic group of China. Nine Y-STR loci were amplified by single multiplex and the PCR products were detected by using ABI Prism(TM) 3100 DNA Sequencer. The allele frequencies and haplotype frequencies at 9 Y-STR loci were determined in a total of 85 unrelated male individuals from Zhuang ethnic group of China. The results indicated that in the 85 unrelated male individuals, except for the DYS426 locus with a low GD value, the GD values for other 8 Y-STR loci ranged from 0.4387 to 0.8129. A total of 70 haplotypes at 9 Y-STR loci were found, the haplotype diversity was 0.9926. It is concluded that the haplotype polymorphism of 9 Y-STR loci are highly polymorphic in Zhuang ethnic group and also significantly different from our previous reported data of unrelated male individnals in southern Chinese Han population.