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.

Randomized controlled trial(RCT) is considered to represent the gold standard for evaluating the efficacy of interventions and has been widely used to evaluate the clinical efficacy of traditional Chinese medicine(TCM). However, there are unique challenges in implementing RCT in TCM. Patients seeking TCM treatment often have preferences for TCM due to the unsatisfactory therapeutic effect of western medicine, their personal intolerance, and their rejection of certain drugs, medical devices, or surgery. Patients are generally reluctant to be randomly assigned to a group, making it challenging to enroll patients in the control group of western medicine during the implementation of RCT in TCM. This has become a prominent problem restricting the implementation of RCT in TCM and needs to be solved urgently. Therefore, this paper introduced commonly used research designs used in solving the problem of enrolling patients in control group during the implementation of RCT in TCM, including Zelen design, partially randomized patient preference trial(PRPP), single-arm objective performance criteria(OPC), cohort studies, single-arm clinical trials using real world data(RWD) alone as the external control group, and the design method based on RWD-augmented control group samples in RCT. The paper outlined the definitions and principles of these methods, evaluated their advantages, disadvantages, and applicable scenarios, and explored their applications in the TCM field, so as to offer insights for solving the difficulty in enrolling patients in the control group during the implementation of RCT in TCM.
Humans ; Medicine, Chinese Traditional/methods* ; Randomized Controlled Trials as Topic/methods* ; Research Design ; Patient Selection ; Drugs, Chinese Herbal/therapeutic use* ; Control Groups

Neuropathic pain, a debilitating condition caused by dysfunction of the somatosensory nervous system, remains difficult to treat due to limited understanding of its molecular mechanisms. Bioinformatics analysis identified cerebellin 2 (CBLN2) as highly enriched in human and murine proprioceptive and nociceptive neurons. We found that CBLN2 expression is persistently upregulated in dorsal root ganglia (DRG) following spinal nerve ligation (SNL) in mice. In addition, transcription factor SOX11 binds to 12 cis-regulatory elements within the Cbln2 promoter to enhance its transcription. SNL also induced SOX11 upregulation, with SOX11 and CBLN2 co-localized in nociceptive neurons. The siRNA-mediated knockdown of Sox11 or Cbln2 attenuated SNL-induced mechanical allodynia and thermal hyperalgesia. High-throughput sequencing of DRG following intrathecal injection of CBLN2 revealed widespread gene expression changes, including upregulation of numerous NF-κB downstream targets. Consistently, CBLN2 activated NF-κB signaling, and inhibition with pyrrolidine dithiocarbamate reduced CBLN2-induced pain hypersensitivity, proinflammatory cytokines and chemokines production, and neuronal hyperexcitability. Together, these findings identified the SOX11/CBLN2/NF-κB axis as a critical mediator of neuropathic pain and a promising target for therapeutic intervention.
Animals ; Neuralgia/metabolism* ; Ganglia, Spinal/metabolism* ; Up-Regulation ; Mice ; NF-kappa B/metabolism* ; SOXC Transcription Factors/genetics* ; Male ; Neuroinflammatory Diseases/metabolism* ; Mice, Inbred C57BL ; Nerve Tissue Proteins/genetics* ; Hyperalgesia/metabolism* ; Signal Transduction ; Spinal Nerves

Objective To observe the value of CT radiomics for differentiating spinal bone islands(BI)and osteoblastic metastases(OBM).Methods Data of 109 BI lesions in 98 patients and 282 OBM lesions in 158 patients(including 103 OBM in 48 lung cancer cases,86 OBM in 52 breast cancer cases and 93 OBM in 58 prostate cancer cases)from 3 medical institutions were retrospectively analyzed.Data obtained from institution 1 were used as the internal dataset and divided into internal training set and internal validation set at a ratio of 7∶3,from institution 2 and 3 were used as external dataset.All datasets were divided into female data subset(including OBM of female lung cancer and breast cancer)and male data subset(including OBM of male lung cancer and prostate cancer).Radiomics features were extracted and screened to construct 3 different support vector machine(SVM)models,including model1 for distinguishing BI and OBM,model2 for differentiating OBM of female lung cancer and breast cancer,and model3 for differentiating OBM of male lung cancer and prostate cancer.Diagnostic efficacy of model1,CT value alone and 3 physicians(A,B,C)for distinguishing BI and OBM were assessed,as well as differentiating efficacy for different OBM of model2 and model3.Receiver operating characteristic(ROC)curves were drawn,and area under the curves(AUC)were calculated and compared.The differential diagnostic efficacy of model2 and model3 were also assessed with ROC analysis and AUC.Results AUC of model1 for distinguishing spinal OBM from BI in internal training set,internal validation set and external dataset was 0.99,0.98 and 0.86,respectively.In internal training set,model1 had higher AUC for distinguishing BI and OBM than that of physician A(AUC=0.78),B(AUC=0.87)and C(AUC=0.93)as well as that of mean CT value(AUC=0.78,all P＜0.05).AUC in internal training set,internal validation set and external dataset of model2 for identifying female lung cancer and breast cancer OBM was 0.79,0.75 and 0.73,respectively,of model3 for discriminating male lung cancer from prostate cancer OBM was 0.77,0.74 and 0.77,respectively.Conclusion CT radiomics SVM model might reliablely distinguish OBM and BI.

Objective:To explore the application value of computed tomography angiography(CTA)combined with neuron-specific enolase(NSE)and insulin-like growth factor-1(IGF-1)in the disease assessment of progressive cerebral infarction.Methods:A total of 110 patients with suspected progressive cerebral infarction admitted to Shanghai Changzheng Hospital from May 2022 to May 2023 were selected as the study subjects,all of them were tested by CTA.According to the deterioration status,63 cases with progressive cerebral infarction were divided into in the study group(progressive cerebral infarction),and then they were further divided into mild degree(19 cases),moderate degree(25 cases)and severe degree(16 cases)according to the condition of neurological deficit of the score of National Institutes of Health Stroke Scale(NIHSS).Other 47 patients with non-progressive cerebral infarction were divided into the control group.The degree of vascular stenosis,the changes of NSE and IGF-1 levels of each group were analyzed,as well as the changes of NSE and IGF-1 levels of neurological deficit with different degrees.The relationship between CTA,NSE and IGF-1 was further explored,and the assessment value of the combined examination of CTA,NSE and IGF-1 on progressive cerebral infarction also was explored.Results:Compared with the control group,the degree of vascular stenosis and NSE levels of the study group increased,while IGF-1 levels of that decreased,and the differences of them between two groups were statistical significance(t=20.893,24.156,40.209,P＜0.05),respectively.Compared with mild patients,IGF-1 levels decreased and NSE levels increased in moderate and severe patients,and the differences were statistical significance(t=4.689,9.103,18.464,23.672,P＜0.05),respectively.Compared with moderate patients,NSE levels increased and IGF-1 levels decreased in severe patients,and the differences were statistical significance(t=5.408,8.118,P＜0.05),respectively.NSE level was positively correlated with the degree of vascular stenosis(r=0.651,P＜0.05),while IGF-1 level was negatively correlated with it(r=-0.617,P＜0.05).Vascular stenosis and NSE were positively correlated with progressive cerebral infarction(r=0.672,P＜0.05),while IGF-1 level was negatively correlated with it(r=-0.629,P＜0.05),respectively.The area under curve(AUC)values of receiver operating characteristic(ROC)curves of CTA,NSE,IGF-1 and the combination of them were respectively 0.688(95%CI:0.594-0.786),0.710(95%CI:0.609-0.811),0.676(95%CI:0.578-0.775)and 0.822(95%CI:0.734-0.910),and the value of the combination of the three indicators was higher in assessing progressive cerebral infarction.Conclusion:The NSE level is higher in patients with progressive cerebral infarction,and the IGF-1 level is lower.The combination of NSE,IGF-1 and CTA can help to enhance the clinical assessment for progressive cerebral infarction,which can provide beneficial basis for clinical diagnosis and treatment of this disease.

Objective To observe the effects of Angelicae Sinensis Radix-Paeoniae Radix alba combined with bone marrow mesenchymal stem cells(BM-MSCs)transplantation on liver inflammation and hepatocytes regeneration in non-alcoholic steatohepatitis(NASH)associated cirrhosis;To discuss its possible mechanism.Methods West diet combined with carbon tetrachloride was used to prepare the NASH related cirrhosis model.The mice were randomly divided into control group,model group,Angelicae Sinensis Radix-Paeoniae Radix alba group,BM-MSCs group,and Angelicae Sinensis Radix-Paeoniae Radix alba+BM-MSCs group,with 12 mice in each group.After successful modeling,corresponding interventions were given according to grouping for 4 consecutive weeks.HE staining was used to observe the morphology of liver tissue;the contents of serum ALT,AST,TBil,TG,ALB,AFP,as well as the contents of IL-1β,TNF-α and HGF in liver tissue were detected;RT-qPCR was used to detect TLR9,MyD88,TRAF6 and NF-κBp65 mRNA expression in liver cells;primary liver cells were separated,CpG ODN 2216 stimulation was induced in vitro,cells supernatant was extracted,and IL-1β and TNF-α content were detected.Results Compared with the control group,inflammatory cell infiltrated in liver tissue of model group mice,accompanied by hepatocyte necrosis and collagen deposition,forming pseudo lobules;the contents of serum ALT,AST,TBil and TG increased,the content of ALB decreased,the content of HGF in liver tissue decreased,the contents of IL-1β and TNF-α increased,with statistical significance(P<0.05);the mRNA expressions of TLR9,MyD88,TRAF6 and NF-κBp65 in liver cells increased(P<0.05),and the contents of IL-1β and TNF-α in cells supernatant increased after CpG ODN 2216 induction(P<0.05).Compared with the model group,the inflammatory infiltration in liver tissue and necrosis of liver cells were reduced and the degree of liver fibrosis was alleviated in the Angelicae Sinensis Radix-Paeoniae Radix alba+BM-MSCs group,the liver tissue damage in Angelicae Sinensis Radix-Paeoniae Radix alba group and BM-MSCs group were slightly improved;except for the TG content in BM-MSCs group,all detection indicators showed significant improvement in each intervention group(P<0.05).Compared with the Angelicae Sinensis Radix-Paeoniae Radix alba group and BM-MSCs group,the Angelicae Sinensis Radix-Paeoniae Radix alba+BM-MSCs group showed statistical significance in related detection indicators(P<0.05),and demonstrated a synergistic effect.Conclusion Angelicae Sinensis Radix-Paeoniae Radix alba combined with BM-MSCs transplantation could effectively improve the liver function and promote liver cell regeneration.The mechanism is associated with the down-regulation of TLR9/NF-κB signaling pathway expressions and function,the inhibition of inflammatory cytokines secretions,and the improvement of liver inflammatory microenviroment.

Objective To optimize the screening methods for antiallergic activity of two Chinese medicines in vitro and compare the antiallergic activity of five medicine pairs in vitro.Methods The degranulation assay of RBL-2H3 cells was optimized,and the activity of the five drugs on inhibiting mast cell degranulation was compared by toluidine blue staining and the release of β-HEX and histamine(HIS).The hyaluronidase inhibition test was optimized to compare the hyaluronidase inhibition effect of five Chinese medicine pairs.Results In the experiment of degranulation of RBL-2H3 cells,5 medicine pairs could inhibit β-HEX release from model cells to different degrees(P<0.05),including Schizonepetae Herba-Saposhnicovia divaricata,Ephedrae Herba-Asarum,Saposhnicovia divaricata-Radix Angelicae dahuricae,Rhizoma Chuanxiong-Asarum.The β-HEX release rate in the supernatant of degranulated model cells was significantly increased(P<0.05)after coculture with Flos Magnoliae-Fructus Xanthii.All the five medicine pairs could reduce HIS release in the supernatant of degranulated model cells.In the hyaluronase inhibition rate test,the hyaluronase inhibition rate of each medicine pair was Rhizoma Chuanxion-Asarum>Saposhnicovia divaricate-Radix Angelicae dahuricae>Schizonepetae Herba-Saposhnicovia divaricate>Ephedrae Herba-Asarum,among which Rhizoma Chuanxiong-Asarum had the strongest anti-allergic activity.Conclusion The trend of antiallergic activity of different drugs obtained by the two methods is consistent in vitro,indicating that the two methods can be used for screening antiallergic activity in vitro.

Objective To explore the effect of circular RNA(circRNA)transcription factor 25(TCF25)-targeting microRNA-128b(miR-128b)on the proliferation and apoptosis of MCF-7 human breast cancer cells.Methods MCF-7 cells were cultured until the loga-rithmic growth stage.Cells were randomly divided into the blank(untransfected),si-NC(transfected si-NC),si-circRNA TCF25(transfected si-circRNA TCF25),pcDNA-circRNA TCF25(transfected pcDNA-circRNA TCF25),miR-NC(transfected miR-NC),miR-128b mimic(transfected miR-128b mimic),miR-128b inhibitor(transfected miR-128b inhibitor),and pcDNA-circRNA TCF25+miR-128b mimic(transfected with pcDNA-circRNA TCF25 and miR-128b mimic)groups.Each group included six multiple pores.Forty-eight hours after transfection,the expression of circRNA TCF25 and miR-128b in each group was determined using real-time reverse transcription-quanti-tative polymerase chain reaction(RT-qPCR).An RNase R enzyme digestion assay was used to identify circular RNA.Subcellular locali-zation of circRNA TCF25 was determined through cytoplasmic-nuclear separation.Cell proliferative activity was measured using the 2,5-diphenyl-2H-tetrazolium bromide(MTT)assay.Apoptosis was detected using flow cytometry.RT-qPCR was performed to determine the mRNA expression levels of phosphatase and tensin homolog(PTEN),proliferating nuclear antigen 67(Ki-67),andcaspase-3.Western blotting was performed to measure PTEN,Ki-67,caspase-3,and cleaved caspase-3 protein expression.The dual-luciferase reporter(DLR)assay was performed to analyze the relationship between circRNA TCF25 and miR-128b.Results Compared to the control group,the relative expression of circRNA TCF25 did not exhibit significant changes after RNase R enzyme treatment(P＞0.05),whereas that of linear TCF25 decreased after RNase R enzyme treatment(P＜0.05).The relative expression of circRNA TCF25 in the cytoplasm was higher than that in the nucleus(P＜0.05).Compared with the blank and si-NC groups,the cell proliferation activity of the si-circRNA TCF25 group decreased;the apoptosis rate increased;Ki-67mRNA and protein expression decreased;and PTEN,caspase-3,and cleaved caspase-3mRNA and protein expression increased.In addition,cell proliferation activity increased and apoptosis rate decreased in the pcDNA-circRNA TCF25 group.Ki-67 mRNA and protein expression were increased,and PTEN,caspase-3,and cleaved caspase-3 mRNA and protein expression decreased,with statistical significance(all P＜0.05).Compared with the blank and miR-NC groups,the cell proliferation activity of the miR-128b mimic group decreased;the apoptosis rate increased;Ki-67mRNA and protein expression decreased;and PTEN,caspase-3,and cleaved caspase-3 mRNA and protein expression were increased,whereas the cell proliferation activity increased and apoptosis rate decreased in the miR-128b inhibitor group.Ki-67mRNA and protein expression were increased,and PTEN,caspase-3,and cleavedcaspase-3mRNA and protein expression decreased,with statistical significance(all P＜0.05).Compared with the pcDNA circRNA TCF25 group,the cell proliferation activity of the pcDNA circRNA TCF25+miR-128b mimic group decreased;the apoptosis rate increased;Ki-67mRNA and protein expression decreased;and PTEN,caspase-3,and cleavedcaspase-3mRNA and protein expression increased,with statistical significance(all P＜0.05).The DLR assay results confirmed that circRNA TCF25 targets miR-128b.Conclusion CircRNAs may play a key role in promoting the proliferation of MCF-7 human breast cancer cells and inhibiting their apoptosis by targeting miR-128b expression;promoting Ki-67 expression;and inhibiting PTEN,caspase-3,and cleaved caspase-3 expression.

Objective To investigate the mechanism of morin-induced autophagy in non-small cell lung cancer A549 cells based on mTOR/STAT3 signaling axis.Methods A549 cells were divided into blank group and 30,60,90,120 and 150 μg·mL-1 of morin groups.After 24,48 and 72 hours of culture,the cell proliferation activity was detected by CCK-8 method,and the cell inhibition rate was calculated.A549 cells were divided into blank group and 30,90,150 μg·mL-1 morin groups.After 14 days of culture,the cell proliferation was detected by colony formation assay.After 24 hours of culture,the cell proliferation ability was detected by BeyoClickTM EdU-488.Apoptosis was detected by flow cytometry;acridine orange staining was used to detect cell autophagy;the formation of autophagosomes was observed by transmission electron microscopy.Western Blot was used to detect the expression levels of apoptosis,autophagy and mTOR/STAT3 signaling axis-related proteins in cells.A549 cells were divided into blank group,blank group + chloroquine(10 μg·mL-1)group,morin(30,150 μg·mL-1)group,morin(30,150 μg·mL-1)+ chloroquine(10 μg·mL-1)group.After 48 hours of intervention,the cell activity was detected by CCK-8 method,and the cell survival rate was calculated.Results Compared with the blank group,the inhibition rate of A549 cells in 60,90,120,150 μ g·mL-1 of morin group was significantly increased after 24 hours of intervention(P＜0.05,P＜0.001).The inhibition rates of A549 cells in 30,60,90,120 and 150 μg·mL-1 of morin groups were significantly increased after 48 and 72 hours of intervention(P＜0.001).The number of A549 cell colonies and the number of green fluorescent proliferation positive cells in the 30,90,150 μg·mL-1 of morin groups were significantly decreased(P＜0.01,P＜0.001),the apoptosis rate was significantly increased(P＜0.01,P＜0.001),and the protein expression level of cleaved-PARP was significantly increased(P＜0.001).The protein expression levels of p-P38/P38 MAPK in A549 cells of 90 and 150 μg·mL-1 of morin groups were significantly increased(P＜0.01,P＜0.001).Different degrees of orange fluorescence appeared in A549 cells of 30,90 and 150 μg·mL-1 of morin groups,and the orange fluorescence of 90 and 150 μg·mL-1 of morin groups was significant.Autophagosomes and autolysosomes appeared in the cytoplasm of A549 cells in 150 μg·mL-1 of morin group,respectively.The protein expression of LC3-Ⅱ in A549 cells of 150 μg·mL-1 of morin group was significantly up-regulated(P＜0.05).The protein expression of Atg16L1-Ⅱ in A549 cells of 90,150 μg·mL-1 of morin group was significantly up-regulated(P＜0.001),and the protein expressions of p-mTOR/mTOR and p-STAT3/STAT3 were significantly down-regulated(P＜0.001).Compared with the morin(150 μg·mL-1)group,the survival rate of A549 cells in the morin(150 μg·mL-1)+chloroquine(10 μg·mL-1)group was significantly increased(P＜0.05).Conclusion Morin can promote the apoptosis of A549 cells and induce autophagy in A549 cells,and the mechanism may be related to mTOR/STAT3 axis.

ObjectiveTo explore the therapeutic effect and mechanism of the Danhe granules on hypercholesterolemia rats by observing the changes in the efficacy indicators and the levels of proteins related to the cholesterol metabolism pathway in the rats under the intervention of Danhe granules. MethodSD rats were randomly assigned to either the blank group or the model group based on their body weight. The blank group had normal chow diets， while the model group was fed high-fat diets for seven weeks. One week after the establishment of the model， the content of the serum total cholesterol （TC） in the model rats was detected. According to the TC value， the model group was further randomly divided into a control group， pravastatin sodium tablet group（4.02 mg·kg^-1）， Xuezhikang capsule group（0.12 g·kg^-1）， high-dose， middle-dose， and low-dose groups of Danhe granules（4.536， 2.268， 1.134 g·kg^-1）. After grouping the model groups， each treatment group received continuous oral gavage for six weeks， with weekly measurements of body weight and food intake （the difference between feed intake and feed surplus）. Six weeks later， the levels of serum TC， triglyceride （TG）， low-density lipoprotein cholesterol （LDL-C）， and high-density lipoprotein cholesterol （HDL-C） were measured. The liver pathology and lipid droplet distribution were evaluated by hematoxylin-eosin （HE） staining and oil red O staining， with scoring and calculation conducted. Rat liver tissue was collected， and western blot and immunohistochemistry （IHC） were used to detect the expression levels of cholesterol metabolism-related proteins namely phosphorylated adenosine 5'-monophosphate （AMP）-activated protein kinase （p-AMPK）， AMPK， 3-hydroxy-3-methyl glutaryl coenzyme A reductase （HMGCR）， low-density lipoprotein receptor （LDLR）， cholesterol 7α-hydroxylase （CYP7A1）， Acyl-coenzyme A： cholesterol acyltransferase 2 （ACAT2）， and apolipoprotein B （ApoB） in hypercholesterolemia rats. ResultCompared with the blank group， the model group showed a significantly higher level of serum TC （P<0.01）. The TG level had no significant change， and the HDL-C level was significantly decreased （P<0.05）. The liver index， steatosis score， total score of pathological state， and the positive area ratio of oil red O staining were significantly increased （P<0.01）， and the protein expression levels of p-AMPK， p-AMPK/AMPK， LDLR， and CYP7A1 were significantly decreased （P<0.05， P<0.01）， while the protein expression levels of AMPK， HMGCR， and ACAT2 were significantly increased （P<0.05， P<0.01）. Compared with the model group， the TC level in each dose group of Danhe granules was significantly decreased （P<0.05）， and the positive area ratio of oil red O staining in the pravastatin sodium tablet group and medium-dose group of Danhe granules was significantly decreased （P<0.05）. In each administration group， the protein expression levels of p-AMPK and p-AMPK/AMPK were significantly increased （P<0.05， P<0.01）， and the levels of HMGCR and ACAT2 were significantly decreased （P<0.01）. The ApoB level showed a downward trend. The CYP7A1 level in the pravastatin sodium tablet group and each dose group of Danhe granules was significantly increased （P<0.05， P<0.01）， and the LDLR level in the pravastatin sodium tablet group， Xuezhikang capsule group， and high-dose and medium-dose groups of Danhe granules was significantly increased （P<0.05， P<0.01）. ConclusionDanhe granules can reduce serum TC levels and improve hepatic steatosis. It may activate AMPK， down-regulate the expression of HMGCR， and inhibit cholesterol synthesis. It can also up-regulate the expression of LDLR and CYP7A1， promote cholesterol uptake and excretion， down-regulate the expression of ACAT2 and ApoB， reduce cholesterol absorption and assembly of LDL and other lipoproteins， and thus play a role in the treatment of hypercholesterolemia.