ObjectiveThis study aimed to observe the impact of sinomenine hydrochloride on the proliferation of fibroblasts and the mRNA expression of related genes in knee joint adhesion and contracture in rabbits. Additionally, we sought to explore its potential mechanisms in combating knee joint adhesion and contracture. MethodsFibroblasts were cultured in vitro, and experimental groups with varying concentrations of sinomenine hydrochloride were established alongside a control group. Cell proliferation was assessed using the CCK-8 assay. Changes in the mRNA expression of fibroblast-related genes following sinomenine hydrochloride treatment were evaluated using RT-qPCR. The impact of the drug on serum levels of inflammatory cytokines was determined using the ELISA method, and the expression of related proteins was assessed using Western blot. ResultsSinomenine hydrochloride was found to inhibit fibroblast viability, with viability decreasing as the concentration of sinomenine hydrochloride increased. The effects of sinomenine hydrochloride in all experimental groups were highly significant (P<0.05). At the mRNA expression level, compared to the control group, sinomenine hydrochloride led to a significant downregulation of inflammatory cytokines in all groups (P<0.05). Additionally, the expression levels of apoptosis-related proteins significantly increased, while Bcl-2 mRNA expression decreased (P<0.05). The mRNA expression levels of the PI3K/mTOR/AKT3 signaling pathway also decreased (P<0.05). At the protein expression level, in comparison to the control group, the levels of inflammatory cytokines IL-6, IL-8, IL-1β, and TGF-β were significantly downregulated in the middle and high-dose sinomenine hydrochloride groups (P<0.05). The expression levels of cleaved-PARP, cleaved caspase-3/7, and Bax increased and were positively correlated with the dose, while the expression levels of the anti-apoptotic protein Bcl-2 and the PI3K/AKT3/mTOR signaling pathway were negatively correlated with the dose. Sinomenine hydrochloride exhibited a significant inhibitory effect on the viability of rabbit knee joint fibroblasts, which may be associated with the downregulation of inflammatory cytokines IL-6, IL-8, and IL-1β, promotion of apoptosis-related proteins cleaved-PARP, cleaved caspase-3/7, and Bax, suppression of Bcl-2 expression, and inhibition of gene expression in the downstream PI3K/AKT3/mTOR signaling pathway. ConclusionSinomenine hydrochloride can inhibit the inflammatory response of fibroblasts in adhesive knee joints and accelerate fibroblast apoptosis. This mechanism may offer a novel approach to improving and treating knee joint adhesion.

Objective:To investigate the clinical features and prognosis of male with anti-melanoma differentiation-associated gene 5 (MDA5) autoantibody.Methods:The clinical data of 246 patients with DM and anti-MDA5 autoantibodies hospitalized by Jiangsu Myositis Cooperation Group from 2017 to 2020 were collected and retrospectively analyzed. Chi-square test was performed to compared between counting data groups; Quantitative data were expressed by M ( Q1, Q3), and rank sum test was used for comparison between groups; Single factor survival analysis was performed by Kaplan-Meier method and Log rank test; Cox regression analysis were used for multivariate survival analysis. Results:①The male group had a higher proportion of rash at the sun exposure area [67.1%(47/70) vs 52.8%(93/176), χ2=4.18, P=0.041] and V-sign [50.0%(35/70) vs 30.7%(54/176), χ2=8.09, P=0.004] than the female group. The male group had higher levels of creatine kinase [112(18, 981)U/L vs 57 (13.6, 1 433)U/L, Z=-3.50, P<0.001] and ferritin [1 500 (166, 32 716)ng/ml vs 569 (18, 14 839)ng/ml, Z=-5.85, P<0.001] than the female group. The proportion of ILD [40.0%(28/70) vs 59.7%(105/176), χ2=7.82, P=0.020] patients and the red blood cell sedimentation rate[31.0(4.0, 101.5)mm/1 h vs 43.4(5.0, 126.5)mm/1 h, Z=-2.22, P=0.026] in the male group was lower than that of the female group, but the proportion of rapidly progressive interstitial lung disease (PR-ILD) [47.1%(33/70) vs 31.3%(55/176), χ2=5.51, P=0.019] was higher than that of the female group. ②In male patients with positive anti-MDA5 antibodies,the death group had a shorter course of disease[1.0(1.0, 3.0) month vs 2.5(0.5,84) month, Z=-3.07, P=0.002], the incidence of arthritis [16.7%(4/24) vs 42.2%(19/45), χ2=4.60, P=0.032] were low than those in survival group,while aspartate aminotransferase (AST)[64(22.1, 565)U/L vs 51(14,601)U/L, Z=-2.42, P=0.016], lactate dehydrogenase (LDH) [485(24,1 464)U/L vs 352(170, 1 213)U/L, Z=-3.38, P=0.001], C-reactive protein (CRP) [11.6(2.9, 61.7) mg/L vs 4.95(0.6, 86.4) mg/L, Z=-1.96, P=0.050], and ferritin levels [2 000(681, 7 676) vs 1 125 (166, 32 716)ng/ml, Z=-3.18, P=0.001] were higher than those in the survival group, and RP-ILD [95.8%(23/24) vs 22.2%(10/45), χ2=33.99, P<0.001] occurred at a significantly higher rate. ③Cox regression analysis indicated that the course of disease LDH level, and RP-ILD were related factors for the prognosis of male anti-MDA5 antibodies [ HR (95% CI)=0.203(0.077, 0.534), P=0.001; HR (95% CI)=1.002(1.001, 1.004), P=0.003; HR (95% CI)=95.674 (10.872, 841.904), P<0.001]. Conclusion:The clinical manifestations of male anti-MDA5 antibody-positive patients are different from those of female. The incidence of ILD is low, but the proportion of PR-ILD is high. The course of disease, serum LDH level, and RP-ILD are prognostic factors of male anti-MDA5 antibody-positive patients.

Objective To investigate the disease spectrum and pathogenic genes of inherited metabolic disorder(IMD)among neonates in Gansu Province of China.Methods A retrospective analysis was conducted on the tandem mass spectrometry data of 286 682 neonates who received IMD screening in Gansu Provincial Maternal and Child Health Hospital from January 2018 to December 2021.A genetic analysis was conducted on the neonates with positive results in tandem mass spectrometry during primary screening and reexamination.Results A total of 23 types of IMD caused by 28 pathogenic genes were found in the 286 682 neonates,and the overall prevalence rate of IMD was 0.63‰(1/1 593),among which phenylketonuria showed the highest prevalence rate of 0.32‰(1/3 083),followed by methylmalonic acidemia(0.11‰,1/8 959)and tetrahydrobiopterin deficiency(0.06‰,1/15 927).In this study,166 variants were identified in the 28 pathogenic genes,with 13 novel variants found in 9 genes.According to American College of Medical Genetics and Genomics guidelines,5 novel variants were classified as pathogenic variants,7 were classified as likely pathogenic variants,and 1 was classified as the variant of uncertain significance.Conclusions This study enriches the database of pathogenic gene variants for IMD and provides basic data for establishing an accurate screening and diagnosis system for IMD in this region.

Since the 18th National Congress of the Communist Party of China(CPC), the CPC and the government have highligh-ted the development of traditional Chinese Medicine(TCM) and issued a series of policies, such as the Plan for Protection and Deve-lopment of Chinese Medicinal Materials(2015-2020) forwarded by the General Office of the State Council in 2015, the Plan for Healthy Development of Traditional Chinese Medicine(2015-2020) released by the General Office of the State Council in the same year, the Healthy China 2030 Plan published by the CPC Central Committee and the State Council in 2016, the Law of the People's Republic of China on Traditional Chinese Medicine which took effect on July 2017, On the Preservation and Innovative Development of Traditional Chinese Medicine promulgated by CPC Central Committee and the State Council in 2019, and Plan for the Development of Traditional Chinese Medicine during the 14th Five-Year Plan Period of China released by the General Office of the State Council in March 2022, to promote the development of the TCM industry, which have brought historical opportunities to the TCM industry. However, TCM industry faces various challenges in the development. In terms of drug development in TCM, the current studies mainly focused on the chemical research and technical requests, which neglected TCM characteristics and cased in conformity between new drug transformation of TCM and clinical practice. Therefore, a more considerable and profound authoritative guideline is needed, and innovative thought and research are necessary for academics and the industry. Through the investigation of the development TCM industry in recent years, this study summarized the policies on and trends of Chinese medicinal materials, new drug development in TCM, catalogue of national basic drugs, and national basic health insurance, and proposed suggestions for further development of TCM industry.
China ; Drugs, Chinese Herbal ; Humans ; Industry ; Medicine, Chinese Traditional ; Policy

Objective:To investigate the expression of hsa_circ_0000437 in the serum of patients with gastric cancer and its clinical value.Methods:The serum samples from 80 patients (57 males and 23 females) with pathologically confirmed gastric cancer (GC), 50 gastric benign disease (28 males and 22 females) and 80 healthy controls (46 males and 34 females) were collected from October 2018 to December 2020 in Affiliated Hospital of Nantong University.Serum samples from 35 of 80 gastric cancer patients after operation were collected. The expression of serum hsa_circ_0000437 was determined by real-time fluorescent quantitative PCR (RT-qPCR). Serum carcinoembryonic antigen (CEA), carbohydrate antigen 199 (CA 199) and carbohydrate antigen 724 (CA724) were determined by chemiluminescence method.Comparisons of serum hsa_circ_0000437 between groups were performed by Mann-Whitney U test.The correlation between serum expression of hsa_circ_0000437 in gastric cancer patients and its clinical pathological characteristics was performed by χ 2 test.Receiver operating characteristic (ROC) curve and the area under the curve of ROC (AUC) were used to evaluate their diagnosis efficiency. Kaplan-Meier survival curve analysis was used to analyze the relationship between the expression level of serum hsa_circ_0000437 and the prognosis of patients. Results:The relative expression of hsa_circ_0000437 in GC, gastric benign disease, healthy controls were 2.252 (1.235, 4.765), 1.598(1.139, 1.982) and 1.000 (0.818, 1.385) respectively.The relative expression of hsa_circ_0000437 in GC was significantly higher than that in gastric benign disease ( P<0.001) and healthy controls ( P<0.001). The difference between gastric benign disease and healthy controls was also statistically significant ( P<0.001).The differences of serum hsa_circ_0000437 expression in GC patients between T stage, N stage, and tumor differentiation were statistically significant. The AUC of hsa_circ_0000437, CEA, CA199 and CA724 in GC patients were 0.863, 0.619, 657 and 0.608 respectively compared with healthy controls. The AUC of above four-parameter panel was 0.892 and the sensitivity was up to 97.5% (78/80). Kaplan-Meier survival curve showed that the overall survival rate of patients with high serum hsa_circ_0000437 expression was significantly lower than that of patients with low expression ( P=0.008). Conclusion:Serum hsa_circ_0000437 could be a biomarker for the auxiliary diagnosis and prognosis of GC.

Objective:To preliminarily estimate and study the effect of nucleic acid testing in blood screening on the residual risk (RR) of transfusion transmitted HBV infection (TTI HBV).Methods:Using the NAT yield/WP ratio model and adopting the relevant data of information management system of practice comparison working party in the Mainland of China, this paper analyzed the trend of the RR of TTI HBV among 18 blood centers from 2015 to 2019 in China, and compared the impact of two kinds of blood screening strategies which were ELISA+ ID-NAT/MP-NAT (individual-donation nucleic acid testing or mini-pool nucleic acid testing) and ELISA + MP-NAT on RR in 2019.Results:The overall trends of the 5-year RR of HBV among 18 blood centers showed by trend chi square test were NAT single positive rate trend χ2= 39.42( P<0.01) and residual risk trend χ2= 279.792( P<0.01); The influence on RR from the differences of ELISA+ ID-NAT/MP-NAT and ELISA+ MP-NAT was statistically significant, and chi square test showed that χ2= 7.4( P<0.01). Conclusions:Since the implementation of nucleic acid testing in the blood screening in China from 2015, the residual risk of transfusion transmitted HBV infection has decreased year by year. The observed two blood screening strategies which dominated in China may lead to discrepancy in the residual risk of TTI.

L_9(3~4) orthogonal experiment design was used to optimize the preparation of the patches,and investigate its affecting factors and skin irritation. Eugenol was taken as the index component to study the release behavior in vitro and percutaneous penetration of Cangai oil transfersomes patches by HPLC.The results showed that the optimal prescription for preparing Cangai oil transfersomes patches were Eudragit E100 0.6 g, succinic acid 0.08 g,triethyl citrate 0.25 g,glycerol 0.2 g.Patches prepared by the preferred preparation had a flat appearance without obvious bubbles.The initial adhesion was 18.33±2.52, the stickiness was(30.01±2.45) min,and the peel strength was(5.62±0.95) kN·m~(-1).The results of affecting factors experiment showed the order of factors affecting its adhesion was humidity>temperature>lighting,and the skin irritation test results showed no significant skin irritation after 24 h of single administration. The results of drug release behavior in vitro showed that the release and the percutaneous penetration of both Cangai oil patches and Cangai oil transfersomes patches conformed to the Higuchi equation.The release amount of eugenol were 80.66% and 82.25% at 72 h, with no significant difference. The cumulative permeation area of eugenol per unit area reached(0.195 6±0.065 9),(0.131 0±0.045 5) mg·cm~(-2) at 72 h, with significant differences(P<0.05).The experiment results proved that the preparation process of Cangai oil transfersomes patches was stable,and the prepared patches had a good adhesion. At the same time,the preparation of transfersomes patches could alleviate and control the release of the drug to a certain extent, and provide a certain experimental basis for clinical pediatric drug safety.
Administration, Cutaneous ; Drug Carriers ; Drug Liberation ; Humans ; Plant Oils/pharmacology* ; Polymethacrylic Acids ; Skin/drug effects* ; Skin Absorption ; Transdermal Patch

Objective To determine the effect of denticleless E3 ubiquitin protein ligase(DTL)on the proliferation and clone formation of multiple myeloma(MM)cells and investigate the related mechanism. Methods Mononuclear cells were extracted from 34 MM patients.Mononuclear cells harvested from 14 healthy volunteers were used as controls.Quantitative polymerase chain reaction was used to detect the change of DTL at mRNA level.Furthermore,12 MM patients and 2 controls were selected,in whom the change of DTL at protein level was detected by Western blot.Human MM cell line RPMI8226 was divided into control(CON)group and DTL-short hairpin RNA(DTL-shRNA)group,which was infected with the CON and DTL-shRNA virus,respectively,for 48 hours.The infection efficiency was detected by using flow cytometry,the knock-down efficiencies at mRNA and protein levels were detected by quantitative polymerase chain reaction and Western blot,the change of cell counts in the next 0,24,48,72,96 hours were measured with CCK8 assay.The CON and DTL-shRNA cells were cultured in semisolid medium.Ten days later,inverted phase microscopy was used to measure the number of colones that contain more than 50 cells,annexin V/propidium iodide double staining to detect apoptosis,and propidium iodide staning to detect cell cycle.Finally,Western blot was empoyed to detect the phosphorylation of P65 and inhibitory subunit-κBα(IκBα)in nuclear factor-κB(NF-κB)pathway and electrophoretic mobility shift assay(EMSA)to detect the NF-κB transcriptional ability. Results The DTL expression was(1.00±0.12)and(9.36±3.71),respectively in the bone marrow mononuclear cells of healthy volunteers and in the CD138+cells of MM patients(t=3.65,P=0.0024).DTL was also highly expressed in MM CD138+positive cells at protein level.After RPMI8226 was infected by CON and DTL-shRNA virus for 48 hours,green fluorescent protein-positive cells accounted for more than 90%.The relative expression of DTL was(1.00±0.01)and(0.21±0.04)(t=33.19,P<0.0001)at mRNA level and(0.52±0.13)and(0.11±0.02)at protein level(t=5.399,P=0.0057).CCK8 revealed that CON and DTL-shRNA cells proliferated by(1.00±0.03)vs.(1.00±0.02),(2.19±0.28)vs.(1.47±0.13),(3.50±0.14)vs.(2.24±0.19),(5.43±0.41)vs.(3.08±0.14),(7.42±0.17)vs.(4.29±013)after 0,24,48,72,and 96 hours(F=24.58,P=0.001).The number of colone containing more than 50 cells was in 76±4 in CON group and 0 in DTL-shRNA group(P<0.01).The proportion of G1 stage cells was(28.61±8.64)% in CON group and(57.25±10.37)% in DTL-shRNA group(t=3.675,P=0.0213).The proportion of annexin V+in CON and DTL-shRNA groups was(3.21±0.89)% vs.(34.71±18.68)%(t=2.895,P=0.0443).After RPMI8226 was infected with CON or DTL-shRNA virus for 48 hours,the relative expression of phosphorylation P65 was(1.52±0.14)vs.(0.82±0.11)(t=6.81,P=0.0024),the P65 relative expression was(0.25±0.04)vs.(0.24±0.08)(t=0.19,P=0.85),the CON and DTL-shRNA phosphorylation-IκBα relative expression was(0.19±0.03)vs.(0.13±0.02)(t=2.882,P=0.0449),and the IκBα was(0.22±0.05)vs.(1.01±0.06)(t=17.52,P<0.0001).Detection of the transcriptional ability of DTL-shRNA NF-κB by EMSA further confirmed the down-regulation of DTL suppressed the NF-κB transcriptional ability. Conclusions DTL is highly expressed in MM cells,and down-regulation of DTL suppresses the cell proliferation,inhibit the colony formation,and induce cell apoptosis and cell cycle arrest.The effect of DTL on the biological functions of MM cells is related to the change of NF-κB pathway.
Apoptosis ; Case-Control Studies ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Multiple Myeloma ; pathology ; NF-kappa B ; metabolism ; Signal Transduction ; Tumor Cells, Cultured ; Ubiquitin-Protein Ligases ; metabolism

Objective To identify the expression of ribosomal protein S9(RPS9)in multiple myeloma(MM)and explore its effect on the biological characteristics of myeloma cells and the corresponding mechanisms. Methods Bone marrow mononuclear cells were harvested in 10 healthy volunteers(CON group)and bone marrow CD138 +cells from 30 MM patients(CD138+group).Quantitative polymerase chain reaction(qPCR)was performed to detect RPS9 expression at mRNA level.In three cases from CON group and 11 cases from CD138+group,Western blot was performed to detect RPS9 at protein level.GSE19784 dataset was employed to detect the relationships of RPS9 expression with the overall survival rate,nuclear factor-κB(NF-κB),small ubiquitin-like modifier(SUMO),and ubiquitin pathway.After the RPS9 knock-down vector was constructed,flow cytometry was performed to detect the infection efficiency and qPCR and Western blot to detect the knock-down efficiency.RPMI8226 was divided into CON group and RPS9-short hairpin RNA(shRNA)group,in which annexin V allophycocyanin/propidium iodide(PI)double staining was performed to detect the change of apoptosis,CCK8 to detect the proliferation change,and PI staining to detect cell cycle change.After sentrin-specific protease 1(SENP1)overexpression vector was constructed,Western blot was performed to detect the phosphorylation of P65 and inhibitory subunit-κBα(IκBα)from NF-κB pathway in CON,RPS9-shRNA,and RPS9-shRNA-SENP1 cells;in addition,annexin V/PI double staining was also performed to detect the apoptosis in these three cells. Results The relative expression of RPS9 in CON group and CD138+group was(1.00±0.12)and(5.45±0.71),respectively(t=4.291,P=0.0036).Western blot showed RPS9 expression was high in most myeloma CD138+cells.The high expression of RPS9 was associated with both extramedullary invasion and overall survival in GSE19784 dataset.After RPMI8226 was infected with CON or RPS9-shRNA lentivirus for 48 hours,flow cytometry confirmed that the infection efficiencies were above 90% in both groups.qPCR and Western blot confirmed that RPS9 expression was inhibited at both mRNA and protein levels.After RPMI8226 CON and RPS9-shRNA infected with virus for 48 hours,the proportion of annexin V-positive cells in CON and RPS9-shRNA cells was(3.47±0.37)% and(18.60±64.00)%(t=9.015,P=0.0008).The proliferation index significantly differed between CON group and RPS9-shRNA group at 72 hours(t=6.846,P=0.0024).When CON and RPS9-shRNA were infected with virus for 48 hours,the proportion of G2 phase cells was(29.28±3.42)% and(10.43±1.43)%,respectively(t=9.329,P=0.0007).The RPS9 expression was positively correlated with SENP1 in GSE19784 dataset and negatively correlated with IκBα coding gene NFKBIA.Western blot further confirmed that RPS9 knockdown inhibited the expression of SENP1,inhibited the phosphorylation of NF-κB subunit P65 and inhibitor IκBα,and promoted the expression of IκBα.Overexpression of SENP1 not only impeded this effect but also reduced RPS9-induced apoptosis. Conclusions RPS9 is highly expressed in MM CD138+cells and is associated with overall survival and extramedullary infiltration.Inhibition of RPS9 can promote apoptosis,cell cycle arrest,and proliferation of myeloma cells.RPS9 can affect the activation of NF-κB pathway and cell apoptosis through SENP1,suggesting that SENP1 may be a key factor in the biological effect of RPS9.
Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Cysteine Endopeptidases ; metabolism ; Humans ; Multiple Myeloma ; metabolism ; Ribosomal Proteins ; metabolism ; Signal Transduction

OBJECTIVE: To clone the promoter sequence of acute monocytic leukemia new antigen gene.MLAA-34 and identify its promoter core region. METHODS: The full-length fragment of MLAA-34 gene promoter region was amplified by PCR, then was ligated into pGL3-Basic vector, and the recombinant plasmid was cloned. Constructed a series of MLAA-34 gene promoter 5' flanking region truncated plasmid. These recombinant plasmids were transfected into U937 and HEK293 cells, and the dual luciferase reporter gene was used to detect the promoter activity of each fragment to determine the minimum active region. Transcription factor binding sites were analyzed by bioinformatics methods. RESULTS: The recombinant plasmid containing MLAA-34 promoter sequence and its truncated plasmid were successfully constructed, and the promoter activity was significantly increased as compared with the empty vector (P＜0.001). The minimal active region of MLAA-34 located between 402 bp and 200 bp. It contained multiple transcription factor binding sites such as E2F1, MZF-1, SP1, USF2 and STAT3. CONCLUSION The promoter of luciferase reporter gene has been successfully constructed with different deletion fragments of MLAA-34, and its core promoter region may contain multiple transcription factor sequence.
Adult ; Antigens, Neoplasm ; genetics ; Apoptosis Regulatory Proteins ; genetics ; Cloning, Molecular ; Genes, Reporter ; HEK293 Cells ; Humans ; Leukemia, Monocytic, Acute ; genetics ; Luciferases ; Promoter Regions, Genetic