Objective: To reveal the molecular biological mechanism of a rare Dc-variant individual using PacBio third-generation sequencing technology. Methods: ABO and Rh blood type identification, DAT, unexpected antibody screening and D antigen enhancement test were conducted by serological testing. The absorption-elution test was used to detect the e antigen. RHCE gene typing was performed by PCR-SSP, and the 1-10 exons of RHCE were sequenced by Sanger sequencing. The full-length sequences of RHCE, RHD and RHAG were detected by PacBio third-generation sequencing technology. Results: Serological findings: Blood type O, Dc-phenotype, DAT negative, unexpected antibody screening negative; enhanced D antigen expression; no detection of e antigen in the absorption-elution test. PCR-SSP genotyping indicated the presence of only the RHCE c allele. Sanger sequencing results: Exons 5-9 of RHCE were deleted, exon 1 had a heterozygous mutation at c. 48G/C, and exon 2 had five heterozygous mutations at c. 150C/T, c. 178C/A, c. 201A/G, c. 203A/G and c. 307C/T. Third-generation sequencing results: RHCE genotype was RHCE 02N. 08/RHCE-D(5-9)-CE; RHD genotype was RHD 01/RHD 01; RHAG genotype was RHAG 01/RHAG 01 (c. 808G>A and c. 861G>A). Conclusion: This Dc-individual carries the allele RHCE 02N. 08 and the novel allele RHCE-D(5-9)-CE. The findings of this study provide data support and a theoretical basis for elucidating the molecular mechanisms underlying RhCE deficiency phenotypes.

In order to investigate whether the effect of Yuxuebi Tablets on the peripheral and central inflammation resolution of mice with inflammatory pain is related to their regulation of G protein-coupled receptor 37(GPR37), an inflammatory pain model was established by injecting complete Freund's adjuvant(CFA) into the paws of mice, with a sham-operated group receiving a similar volume of normal saline. The mice were assigned randomly to the sham-operated group, model group, ibuprofen group(91 mg·kg~(-1)), and low-, medium-, and high-dose groups of Yuxuebi Tablets(60, 120, and 240 mg·kg~(-1)). The drug was administered orally from days 1 to 19 after modeling. Von Frey method and the hot plate test were used to detect mechanical pain thresholds and heat hyperalgesia. The levels of interleukin-10(IL-10) and transforming growth factor-beta(TGF-β) in the spinal cord were quantified using enzyme-linked immunosorbent assay(ELISA), and the mRNA and protein expression of GPR37 in the spinal cord was measured by real-time quantitative reverse transcription PCR(qRT-PCR) and Western blot. Additionally, immunofluorescence was used to detect the expression of macrosialin antigen(CD68), mannose receptor(MRC1 or CD206), and GPR37 in dorsal root ganglia, as well as the expression of calcium-binding adapter molecule 1(IBA1), CD206, and GPR37 in the dorsal horn of the spinal cord. The results showed that compared with those of the sham-operated group, the mechanical pain thresholds and hot withdrawal latency of the model group significantly declined, and the expression of CD68 in the dorsal root ganglia and the expression of IBA1 in the dorsal horn of the spinal cord significantly increased. The expression of CD206 and GPR37 significantly decreased in the dorsal root ganglion and dorsal horn of the spinal cord, and IL-10 and TGF-β levels in the spinal cord were significantly decreased. Compared with those of the model group, the mechanical pain thresholds and hot withdrawal latency of the high-dose group of Yuxuebi Tablets significantly increased, and the expression of CD68 in the dorsal root ganglion and IBA1 in the dorsal horn of the spinal cord significantly decreased. The expression of CD206 and GPR37 in the dorsal root ganglion and dorsal horn of the spinal cord significantly increased, as well as IL-10 and TGF-β levels in the spinal cord. These findings indicated that Yuxuebi Tablets may reduce macrophage(microglial) infiltration and foster M2 macrophage polarization by enhancing GPR37 expression in the dorsal root ganglia and dorsal horn of the spinal cord of CFA-induced mice, so as to improve IL-10 and TGF-β levels, promote resolution of both peripheral and central inflammation, and play analgesic effects.
Inflammation/genetics* ; Pain/genetics* ; Drugs, Chinese Herbal/administration & dosage* ; Animals ; Mice ; Freund's Adjuvant/pharmacology* ; Ibuprofen ; Pain Threshold/drug effects* ; Hyperalgesia/genetics* ; Ganglia, Spinal ; Interleukin-10/genetics* ; Transforming Growth Factor beta/genetics* ; Reverse Transcriptase Polymerase Chain Reaction ; Tablets ; Receptors, G-Protein-Coupled

OBJECTIVE: To investigate the regulatory effects of two traditional mineral medicines (TMMs), Gypsum Fibrosum (Shigao, GF) and Terra Flava Usta (Zaoxintu, TFU), on gut-beneficial bacteria in mice, and preliminarily explore their mechanisms of action. METHODS: Mice were randomly divided into 3 groups (n=10 per group): the control group (standard diet), the GF group (diet supplemented with 2% GF), and the TFU group (diet supplemented with 2% TFU). After 4-week intervention, 16S rRNA gene sequencing was used to analyze the changes in the gut microbiota (GM). Scanning electron microscopy, in combination with coumarin A tetramethyl rhodamine conjugate and Hoechst stainings, was used to observe the bacteria and biofilm formation. RESULTS: Principal coordinate analysis revealed that GF and TFU significantly altered the GM composition in mice. Further analysis revealed that GF and TFU affected different types of gut bacteria, suggesting that different TMMs may selectively modulate specific bacterial populations. For certain bacteria, such as Faecalibaculum and Ileibacterium, both GF and TFU exhibited growth-promoting effects, implying that they may be sensitive to TMMs and that different TMMs can increase their abundance through their respective mechanisms. Notably, Lactobacillus reuteri, a widely recognized and used probiotic, was significantly enriched in the GF group. Random forest analysis identified Ileibacterium valens as a potential indicator bacterium for TMMs' impact on GM. Further mechanistic studies showed that gut bacteria formed biofilm structures on the TFU surface. CONCLUSIONS This study provides new insights into the interaction between TMMs and GM. As safe and effective natural clays, GF and TFU hold promise as potential candidates for prebiotic development.
Animals ; Gastrointestinal Microbiome/drug effects* ; Bacteria/growth & development* ; Mice ; Biofilms/drug effects* ; Male ; RNA, Ribosomal, 16S/genetics*

Disruption of the intestinal mucosal barrier caused by gut dysbiosis and metabolic imbalance is the underlying pathology of inflammatory bowel disease (IBD). Traditional Chinese medicine Wuji Wan (WJW) is commonly used to treat digestive system disorders and showed therapeutic potential for IBD. In this interdisciplinary study, we aim to investigate the pharmacological effects of WJW against experimental colitis by combining functional metabolomics and gut-microbiota sequencing techniques. Treatment with WJW altered the profile of the intestinal microbiota and notably increased the abundance of Lactobacillus, thereby facilitating the conversion of tryptophan into indole-3-acetic acid (IAA) and indoleacrylic acid (IA). These indole derivatives activated the aryl hydrocarbon receptor (AhR) pathway, which reduced colonic inflammation and restored the expression of intestinal barrier proteins. Interestingly, the beneficial effects of WJW on gut barrier function improvement and tryptophan metabolism were disappeared in the absence of gut microbiota. Finally, pre-treatment with the AhR antagonist CH-223191 confirmed the essential role of IAA-mediated AhR activation in the therapeutic effects of WJW. Overall, WJW enhanced intestinal barrier function and reduced colonic inflammation in a murine colitis model by modulating Lactobacillus-IAA-AhR signaling pathway. This study provides novel insights into colitis pathogenesis and presents an effective therapeutic and preventive approach against IBD.

OBJECTIVE: Varied acupoint selections represent a potential cause of the uncertainty surrounding the efficacy of acupuncture for knee osteoarthritis (OA). Skin temperature, a guiding factor for acupoint selection, may help to address this issue. This study explored thermal sensitization of acupoints used for the treatment of knee OA. METHODS: This cross-sectional case-control study enrolled cases aged 45-75 years with symptomatic knee OA and age- and gender-matched non-knee OA controls in a 1:1 ratio. All participants underwent infrared thermographic imaging. The primary outcome was the relative skin temperature of acupoint (STA), and the secondary outcome was the absolute STA of 11 acupoints. The Z test was used to compare the relative and absolute STAs between the groups. Principal component analysis was used to extract the common factors (CFs, acupoint cluster) in the STAs. A general linear model was used to identify factors affecting the STA in the knee OA cases. For the group comparisons of relative STA, P < 0.0045 (adjusted for 11 acupoints through Bonferroni correction) was considered to indicate statistical significance. For other analyses, P < 0.05 was used as the threshold for statistical significance. RESULTS: The analysis included 308 participants, consisting of 151 cases (mean age: [64.58 ± 6.67] years; male: 25.83%; mean body mass index: [25.70 ± 3.16] kg/m²) and 157 controls (mean age: [63.37 ± 5.96] years; male: 26.11%; mean body mass index: [24.47 ± 2.84] kg/m²). The relative STAs of ST34 (P = 0.0001), EX-LE2 (P < 0.0001), EX-LE5 (P = 0.0006), SP10 (P < 0.0001), BL40 (P = 0.0012) and GB39 (P = 0.0037) were higher in the knee OA group. No difference was found in the STAs of ST35, ST36, SP9, GB33 and GB34. Four CFs were identified for relative STA in both groups. The acupoints within each CF were consistent between the groups. The mean values of the relative STAs across each CF were higher in the knee OA group. In the knee OA cases, no factors were observed to affect the relative STA, while age and gender were found to affect the absolute STA. CONCLUSION Among patients with knee OA, thermal sensitization occurs in the acupoints of the lower extremity, exhibiting localized and regional thermal consistencies. The thermally sensitized acupoints that we identified in this study, ST34, SP10, EX-LE2, EX-LE5, GB39 and BL40, may be good choices for the acupuncture treatment of knee OA. Please cite this article as: Tu JF, Wang XZ, Yan SY, Wang YR, Yang JW, Shi GX, Zhang WZ, Jing LN, Yang LS, Liu DH, Wang LQ, Mi BH. Thermal sensitization of acupoints in patients with knee osteoarthritis: A cross-sectional case-control study. J Integr Med. 2025; 23(3): 289-296.
Humans ; Osteoarthritis, Knee/physiopathology* ; Male ; Cross-Sectional Studies ; Middle Aged ; Female ; Acupuncture Points ; Case-Control Studies ; Aged ; Skin Temperature ; Acupuncture Therapy

OBJECTIVE: CRISPR-Cas protects bacteria from exogenous DNA invasion and is associated with bacterial biofilm formation and pathogenicity. METHODS: We analyzed the type I-F CRISPR-Cas system of Legionella pneumophila WX48, including Cas1, Cas2-Cas3, Csy1, Csy2, Csy3, and Cas6f, along with downstream CRISPR arrays. We explored the effects of the CRISPR-Cas system on the in vitro growth, biofilm-forming ability, and pathogenicity of L. pneumophila through constructing gene deletion mutants. RESULTS: The type I-F CRISPR-Cas system did not affect the in vitro growth of wild-type or mutant strains. The biofilm formation and intracellular proliferation of the mutant strains were weaker than those of the wild type owing to the regulation of type IV pili and Dot/Icm type IV secretion systems. In particular, Cas6f deletion strongly inhibited these processes. CONCLUSION The type I-F CRISPR-Cas system may reduce biofilm formation and intracellular proliferation in L. pneumophila.
Legionella pneumophila/pathogenicity* ; CRISPR-Cas Systems ; Biofilms/growth & development* ; Phenotype ; Bacterial Proteins/metabolism* ; Gene Deletion

Objective To explore the therapeutic material basis and antitussive mechanism of Ganke Formula.Methods Ultra-high performance liquid chromatography-quadrupole-time of flight mass spectrometry(UPLC-Q-TOF/MS)technology was used to analyze and identify the components of Ganke Formula in serum.30 rats were randomly divided into control group,model group,experimental-L group,experimental-M group and experimental-H group,6 rats per group.Acute bronchitis rat model was established by smoke inhalation and cold stimulus.The experimental-L,-M,-H groups were respectively given 1.86,4.66,9.32 g·kg-1(crude drug/weight)dose of Ganke Formula per day by intragastric administration for 7 days.The control group and model group were given the equal amount of normal saline.The contents of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in rat lung tissue were determined by enzyme-linked immunosorbent assay.The expressions of phosphatidylinositol 3-kinase(PI3K)and phosphorylated protein kinase B(p-Akt)in lung were detected by Western blotting.Results Twenty-six absorbed components have been identified by UPLC-Q-TOF/MS.The contents of IL-1β in control group,model group,experimental-M,-H group were(57.80±7.67),(186.48±8.50),(166.05±9.90)and(143.19±6.31)pg·mL-1;TNF-α contents were(47.14±8.55),(316.22±9.49),(68.93±7.94)and(65.93±7.10)pg·mL-1;the relative expression levels of PI3K in lung tissues were 0.38±0.05,0.97±0.10,0.68±0.15 and 0.56±0.10;the relative expression levels of p-Akt were 0.34±0.14,0.93±0.05,0.63±0.16 and 0.49±0.14,respectively.Compared with the model group,the above indicators in the experimental-H group and control group were statistically different(P＜0.01 or P＜0.05).Conclusion Ganke Formula can intervene in the expression of inflammatory and immune regulatory factors by regulating PI3K/Akt signaling pathway,improve airway inflammation,and thus exert cough relieving effects.

Objective To investigate the role of long non-coding RNA(lnc RNA)ZFAS1 in glioma cells'sensitivity to cisplatin and its underlying mechanisms.Methods By analyzing the knockdown of ZFAS1 on the sensitivity of glioma cells to cisplatin using real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)experiments,and the cells were divided into sh-NC group(transfected with sh-NC lentiviral plasmid),sh#1 group(transfected with sh-ZFAS1-1 lentiviral plasmid)and sh#2 group(transfected with sh-ZFAS1-2 lentiviral plasmid).Dual luciferase experiments verified the interaction between ZFAS1 and miR-193b-3p,and the cells were divided into ZFAS1-WT+NC inhibitor group(transfected with ZFAS1 wild-type plasmid and NC inhibitor),ZFAS1-WT+miR-193b-3p inhibitor group(transfected with ZFAS1 wild-type plasmid and miR-193b-3p inhibitor),ZFAS1-Mut+NC inhibitor group(transfected with ZFAS1 mutant plasmid and NC inhibitor)and ZFAS1-Mut+miR-193b-3p inhibitor group(transfected with ZFAS1 mutant plasmid and miR-193b-3p inhibitor).Cell counting kit-8(CCK-8)and terminal deoxynucleotidly transferase mediated labeling(TUNEL)experiments were used to analyze the effect of ZFAS1/miR-193b-3p on the sensitivity of glioma cells to cisplatin,and the cells were divided into blank control group(0 μg·mL-1 cisplatin treatment of U251 cells),0.5 μg·mL-1 cisplatin+sh-NC+NC inhibitor group(0.5 μg·mL-1 cisplatin treatment of U251 cells co-transfected with sh-NC lentiviral plasmid and NC inhibitor),0.5 μg·mL-1 cisplatin+sh#1+NC inhibitor group(0.5 μg·mL-1cisplatin treatment of U251 cells co-transfected with sh-NC lentiviral plasmid and NC inhibitor),and 0.5 μg·mL-1 cisplatin+sh#1+miR-193b-3p inhibitor group(0.5 μg·mL-1 cisplatin treatment of U251 cells co-transfected with sh-ZFAS1-1 lentiviral plasmid and miR-193b-3p inhibitor).Results The results of the experiment showed that the expression levels of ZFAS1 in the sh-NC group,sh#1 group and sh#2 group were 1.00±0.17,0.48±0.06 and 0.68±0.08.The fluorescence activities of ZFAS 1-WT+NC inhibitor group,ZFAS1-WT+miR-193b-3p inhibitor group,ZFAS1-Mut+NC inhibitor group and ZFAS1-Mut+miR-193b-3p inhibitor group were 1.00±0.10,1.45±0.11,1.02±0.09 and 0.97±0.13.The proliferation rates at 72 h for the blank control group,0.5 μg·mL-1 cisplatin+sh-NC+NC inhibitor group,0.5 μg·mL-1 cisplatin+sh#1+NC inhibitor group and 0.5 μg·mL-1cisplatin+sh# 1+miR-193b-3p inhibitor group were(100.00±14.13)％,(96.62±9.82)％,(60.56±6.08)％and(78.64±7.22)％;while the apoptosis rates at 72 h were(9.52±1.11)％,(10.12±1.34)％,(16.08±1.52)％and(12.22±1.19)％.Comparied between blank control group and 0.5 μg·mL-1 cisplatin+sh-NC+NC inhibitor group,0.5 μg·mL-1 cisplatin+sh#1+NC inhibitor group and 0.5 μg·mL-1 cisplatin+sh # 1+miR-193b-3p inhibitor group,the differences were statistically significant(all P＜0.05).Conclusion This study reveals the important role of ZFAS1 in cisplatin sensitivity in glioma and elucidates its mechanism of influencing drug sensitivity through the regulation of miR-193b-3p.

The present study identified chemical constituents of Honghua Xiaoyao Tablet (HXT) and explored its biological connotation and characteristics on the premenstrual syndrome (PMS) treatment from the "disease-syndrome-symptom" association network. UHPLC-Q Exactive Orbitrap HRMS technology was applied to analyze the chemical constituents in HXT. According to the composition principles, the compatible herbs of HXT were divided into the Shugan Jieyu group, Huoxue Tiaojing group and Yiqi Jianpi group. The candidate targets of the corresponding prescriptions of HXT efficacy groups were collected from the Pharmmapper database and Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine (TCMIP) v2.0. The gene set related to the clinical symptoms included in Traditional Chinese and Western Medicine diagnosis and treatment standards were obtained from SoFDA, GeneCards, DisGeNET, MalaCards and literature published. The "HXT candidate targets-PMS (liver depression, Qi stagnation, and blood stasis syndrome) genes" network was constructed based on the gene interaction information, and further, the core network targets were screened out by topological characteristics of calculating network, and the functional exploration was carried out based on Kyoto Encyclopedia of Genes and Genomes (KEGG) for exploring the therapeutic advantages in PMS treatment of HXT efficacy groups, which were further verified experimentally in vitro. A total of 109 components from HXT were identified, including 20 components from Shugan Jieyu group enriched in the neurological system, estrogen regulation, and "immune-inflammation" related pathways, 77 components from Huoxue Tiaojing group enriched in the blood-circulation system, "immune-inflammation" and estrogen regulation related pathways and 30 components from Yiqi Jianpi group regulating immune inflammation, digestive system, and hormone levels. The biochemical indicator detection demonstrated that both the levels of 5-HT and DA in the hypothalamus tissues and the levels of E₂, NO, VEGF and RLN in the uterine tissues of PMS model rats were lower than those in controls, which the levels of OT, PROG, IL-6, IL-1β and TNF-α in the uterine tissues were increased in PMS group, which were all reversed by the administration of HXT, indicating that this prescription may regulate the synthesis and secretion of estrogen, intervene in neurotransmitter synthesis and signal transduction, reverse the imbalance of "immune-inflammation", and regulate digestive system function through various biological pathways, exerting the liver-smoothing, Qi-regulating, blood-activating and spleen-tonifying comprehensive effects, leading to alleviating the "neuroendocrine-endocrine-immune" system and blood-circulation disorders. The relevant results may provide a reference for clarifying the advantages and efficacy of HXT in treating PMS with liver stagnation, Qi stagnation, and blood stasis syndrome, and exploring its therapeutic advantages. The animal experiment of this study was approved by the Ethics Committee of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences (approval number: 2023B248).

This study aimed to investigate the analgesic effect of chlorogenic acid on cisplatin-induced neuropathic pain and explored the underlying molecular mechanisms. The animal experimental protocol has been reviewed and approved by Laboratory Animal Ethics Committee of Xinxiang Central Hospital, in compliance with the Institutional Animal Care Guidelines. Von Frey hair and a radiant heat was employed to measure mechanical allodynia and thermal hyperalgesia; Western blot was used to examine transient receptor potential vanilloid type-1 (TRPV1) protein expression in the rat dorsal root ganglion (DRG); patch clamp was used to record TRPV1 currents in DRG neurons. The experimental results showed that chlorogenic acid could attenuate cisplatin-induce mechanical allodynia and thermal hyperalgesia in rats. The expression of TRPV1 protein in DRGs was increased in cisplatin-treated rats, while chlorogenic acid also could reverse cisplatin-induced the upregulation of TRPV1 protein. Forthermore, chlorogenic acid could attenuate cisplatin-mediated the upregulation of TRPV1 current density. These above results indicated that chlorogenic acid could alleviate cisplatin-induced pain hypersensitivity through inhibition of the expression and function of TRPV1 in rats.