OBJECTIVE To explore the mechanism of joint injection of Caulophyllum robustum Maxim in the treatment of rheumatoid arthritis （RA）. METHODS The targets of main saponins in C. robustum Maxim were obtained from Swiss Target Prediction， and the RA treatment targets collected from the GeneCards and OMIM database were intercrossed to establish an interaction network based on network pharmacology. Gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway analysis were performed. RA model was established by injecting complete Freund’s adjuvant into the back of rabbits for verification. The arthritis index score， knee diameter and pain threshold of rabbits were compared. Pathological examination of rabbit synovial tissue was carried out. The levels of tumor necrosis factor α （TNF-α）， interleukin-1β （IL-1β） and IL-6 in rabbit serum and synovial fluid were detected. The phosphorylation levels of tyrosine protein Janus kinase 2 （JAK2） and signal transducer and activator of transcription 3 （STAT3） proteins in rabbit synovium were detected. RESULTS Network pharmacology identified 143 intersection targets between the drug and RA. After the construction of the “drug-component-target” network， the core components of the network were echinocystic acid， oleanolic acid， hederagenin， cauloside A and cauloside C， etc. Additionally， the top 10 core targets of PPI network were SRC， STAT3， MAPK1， EGFR， PIK3CA， MAPK3， GRB2， JUN， PTPN11 and JAK2. The results of KEGG pathway analysis showed that the JAK/STAT signaling pathway was mainly involved in the treatment of RA by joint injection of C. robustum Maxim. Results of validation test showed that compared with model group， joint injection of C. robustum Maxim could reduce the swelling of rabbit knee joint， relieve the hyperplasia of synovial layer， reduce the hyperplasia of lower connective tissue， and reduce the number of inflammatory cells and capillaries. The arthritis index score （excluding low-dose group of C. robustum Maxim）， knee diameter， the levels of TNF-α， IL-1β and IL-6 in serum and synovial fluid， and the protein phosphorylation levels of JAK2 and STAT3 were decreased significantly （P＜0.05 of P＜0.01）， while the pain threshold were reduced significantly （P＜0.01）. CONCLUSIONS The core components that may alleviate the inflammatory response of RA in joint injection of C. robustum Maxim could include echinocystic acid， oleanolic acid， hederagenin， cauloside A， and cauloside C. Its mechanism may be related to the inhibition of JAK/STAT signaling pathway and the reduction of inflammatory responses.

ObjectiveTo investigate the potential mechanism of action of the Qufeng Tongqiao Cough-relieving Decoction （祛风通窍止咳方， QTCD） in the treatment of upper airway cough syndrome （UACS）. MethodsTwenty-four guinea pigs were randomly divided into blank group， model group， traditional Chinese medicine （TCM） group， and inhibitor group， with six guinea pigs in each group. Except for the blank group， guinea pigs were sensitized with ovalbumin and aluminum hydroxide via intraperitoneal injection， followed by ovalbumin nasal drops combined with smoke exposure to establish the UACS model. After modeling， the TCM group was administered QTCD 0.9 g/（100 g·d） by gavage， the inhibitor group received the transient receptor potential vanilloid receptor 4 （TRPV4） inhibitor GSK2193874 1 mmol/L， 5 min by nebulisation， and the blank group and model group were given 2 ml/（100 g·d） normal saline by gavage once daily. After 7 days of treatment， a cough provocation test was performed using 0.4 mol/L citric acid. The levels of IgE in serum and inflammatory cytokines， including interleukin-6 （IL-6）， interleukin-8 （IL-8） in serum， bronchoalveolar lavage fluid （BALF）， and nasal lavage fluid （NLF） were detected by enzyme-linked immunosorbent assay （ELISA）. Histopathological changes in lung and nasal mucosal tissues were observed by hematoxylin-eosin （HE） staining. Immunohistochemistry was used to detect the protein levels of TRPV4， substance P （SP）， and calcitonin gene-related peptide （CGRP） in lung and nasal mucosal tissues. Real-time polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of TRPV4， SP， and CGRP in lung tissues. ResultsHE staining showed significant structural damage and infiltration of inflammatory cells in the lung and nasal mucosal tissues in the model group， while the TCM group and inhibitor group showed improved pathological changes. Compared with the blank group， the model group showed increased cough frequency， serum IgE level， and IL-6 and IL-8 levels in serum， BALF， and NLF. The protein levels of TRPV4， SP， and CGRP in lung and nasal mucosal tissues and their mRNA expression were elevated （P＜0.05 or P＜0.01）. Compared with the model group， the TCM group and inhibitor group showed reduced cough frequency， serum IgE level， and TRPV4 and SP mRNA expression in lung tissues. The TCM group showed reduced IL-6 and IL-8 levels in serum， BALF， and NLF， and reduced TRPV4 and CGRP protein levels in lung and nasal mucosal tissues. The inhibitor group showed reduced IL-6 and IL-8 levels in serum， BALF， and NLF， reduced IL-6 in BALF， reduced IL-8 in NLF， and decreased TRPV4， SP， and CGRP protein levels in lung tissues and SP and CGRP protein levels in nasal mucosal tissues （P＜0.05 or P＜0.01）. Compared with the TCM group， the inhibitor group had increased serum IgE， IL-6， and IL-8 levels， increased IL-6 level in BALF， and increased IL-8 levle in NLF， but decreased SP protein level in lung tissues and increased TRPV4 and SP mRNA expression in lung tissues （P＜0.01）. ConclusionQTCD effectively reduces cough frequency in the UACS guinea pig model. Its mechanism may involve inhibiting the activation of the TRPV4 pathway， improving airway neurogenic inflammation， alleviating inflammatory responses， and reducing cough hypersensitivity.

Although metal blocks have been widely used for reconstructing uncontained tibial bone defects, the influence of their elastic modulus on the stability of tibial prosthesis fixation remains unclear. Based on this, a finite element model incorporating constrained condylar knee (CCK) prosthesis, tibia, and metal block was established. Considering the influence of the post-restraint structure of the prosthesis, the effects of variations in the elastic modulus of the block on the von Mises stress distribution in the tibia and the block, as well as on the micromotion at the bone-prosthesis fixation interface, were investigated. Results demonstrated that collision between the insert post and femoral prosthesis during tibial internal rotation increased tibial von Mises stress, significantly influencing the prediction of block elastic modulus variation. A decrease in the elastic modulus of the metal block resulted in increased von Mises stress in the proximal tibia, significantly reduced von Mises stress in the distal tibia, decreased von Mises stress of the block, and increased micromotion at the bone-prosthesis fixation interface. When the elastic modulus of the metal block fell below that of bone cement, inadequate block support substantially increased the risk of stress shielding in the distal tibia and fixation interface loosening. Therefore, this study recommends that biomechanical investigations of CCK prostheses must consider the post-constraint effect, and the elastic modulus of metal blocks for bone reconstruction should not be lower than 3 600 MPa.
Knee Prosthesis ; Humans ; Finite Element Analysis ; Tibia/surgery* ; Elastic Modulus ; Arthroplasty, Replacement, Knee/methods* ; Stress, Mechanical ; Metals ; Prosthesis Design ; Knee Joint/surgery* ; Biomechanical Phenomena

Objective To investigate the effect of Mcart1 knockout on acute inflammation of macrophages in vitro and in vivo.Methods The Mcart1 knockout mice were used to establish a sepsis model induced by lipopolysaccharide(LPS),and the survival period was measured.Bone marrow derived macrophages(BMDM)of LPS-induced inflam-mation mice were cultured in DMEM high-glucose medium.The mRNA levels of M1 and M2 related cytokines of BM-DM after LPS stimulation were detected by RT-qPCR.The expression level of inflammation-related cytokines in serum of sepsis mice was detected by ELISA.Results Compared with wild type mice(Mcart1flox/flox),the survival time length of sepsis in Mcart1 knockout mice(Mcart1Lyz2-Cre)was significantly shortened(P＜0.001).After inflammation,the mRNA level of M1-related cytokines was up-regulated in BMDM cells of Mcart1Lyz2-Cre mice(P＜0.05);The mRNA level of M1-related cytokines was down-regulated(P＜0.05);The expression of M1-related mediators in serum of sep-sis Mcart1Lyz2-Cre mice was up-regulated(P＜0.01).Conclusions Mcart1 knockout can significantly raise the inflam-matory response of macrophages and aggravate the pathological symptoms of sepsis mice.

Benzylisoquinoline alkaloids (BIAs) are a structurally diverse group of plant metabolites renowned for their pharmacological properties. However, sustainable sources for these compounds remain limited. Consequently, researchers are focusing on elucidating BIA biosynthetic pathways and genes to explore alternative sources using synthetic biology approaches. CYP80B, a family of cytochrome P450 (CYP450) enzymes, plays a crucial role in BIA biosynthesis. Previously reported CYP80Bs are known to catalyze the 3'-hydroxylation of (S)-N-methylcoclaurine, with the N-methyl group essential for catalytic activity. In this study, we successfully cloned a full-length CYP80B gene (StCYP80B) from Stephania tetrandra (S. tetrandra) and identified its function using a yeast heterologous expression system. Both in vivo yeast feeding and in vitro enzyme analysis demonstrated that StCYP80B could catalyze N-methylcoclaurine and coclaurine into their respective 3'-hydroxylated products. Notably, StCYP80B exhibited an expanded substrate selectivity compared to previously reported wild-type CYP80Bs, as it did not require an N-methyl group for hydroxylase activity. Furthermore, StCYP80B displayed a clear preference for the (S)-configuration. Co-expression of StCYP80B with the CYP450 reductases (CPRs, StCPR1, and StCPR2), also cloned from S. tetrandra, significantly enhanced the catalytic activity towards (S)-coclaurine. Site-directed mutagenesis of StCYP80B revealed that the residue H205 is crucial for coclaurine catalysis. Additionally, StCYP80B exhibited tissue-specific expression in plants. This study provides new genetic resources for the biosynthesis of BIAs and further elucidates their synthetic pathway in natural plant systems.
Cytochrome P-450 Enzyme System/chemistry* ; Benzylisoquinolines/chemistry* ; Hydroxylation ; Plant Proteins/chemistry* ; Alkaloids/metabolism* ; Stephania tetrandra/genetics*

Objective:To investigate the effects of Juglone on the apoptosis of osteosarcoma(OS)cells(U2OS and MG63 cells)through the cysteinyl aspartate specific proteinase-3(Caspase-3)/gasdermin E(GSDME)-mediated pyroptosis pathway.Methods:The U2OS and MG63 cells were cultured in vitro and divided into control group,different concentrations(5,10 and 20 μmol·L-1)of Juglone groups and Caspase-3 inhibitor Z-DEVD-FMK group(10 μmol·L-1 Juglone+30 μmol·L-1 Z-DEVD-FMK).The survival rates of cells in various groups were assessed by cell counting kit-8(CCK-8)assay,and the apoptotic rates were detected by flow cytometry.Lactate dehydrogenase(LDH)release assay was used to measure the release rates of LDH from the cells.Western blotting method was used to detect the expression levels of apoptosis-related proteins including B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),cleaved-Caspase-3 and poly(ADP-ribose)polymerase(PARP)and pyroptosis-related proteins including GSDME full form(GSDME-F)and GSDME N-terminal(GSDME-N).The levels of interleukin-1β(IL-1β)and interleukin-18(IL-18)in the cell supernataut in various groups were measured by enzyme-linked immunosorbent assay(ELISA)method.Results:Compared with control group,the survival rates of cells in 5,10,and 20 μmol·L-1Juglone groups were significantly decreased(P＜0.05 or P＜0.01),and the 50％inhibitory concentration(IC50)values of U2OS cells and MG63 cells were 8.4 and 10.2 μmol·L-1,respectively.Compared with control group,the apoptotic rates and LDH release rates of U2OS and MG63 cells in 5 and 10 μmol·L-1Juglone groups were significantly increased(P＜0.05 or P＜0.01).Compared with control group,the expression levels of Bax,cleaved-Caspase-3,and cleaved-PARP proteins in 5 and 10 μmol·L-1 Juglone groups were significantly increased(P＜0.01),while the expression levels of Bcl-2 protein were significantly decreased(P＜0.01).Compared with control group,the levels of IL-1β and IL-18 in cell supernatant in 5 and 10 μmol·L-1Juglone groups were increased(P＜0.01).Compared with control group,the expression levels of cleaved-Caspase-3 and GSDME-N proteins in 5 and 10 μmol·L-1 Juglone groups were significantly increased(P＜0.01),while there was no difference in the expression level of GSDME-F protein(P＞0.05).Compared with 10 μmol·L-1 Juglone group,the expression levels of cleaved-Caspase-3 and GSDME-N in Z-DEVD-FMK group were significantly decreased(P＜0.01),while there was no difference in the expression level of GSDME-F protein(P＞0.05).Conclusion:Juglone can induce the apoptosis of U2OS and MG63 cells and cause the Caspase-3/GSDME-mediated pyroptosis.

Objective:To discuss the effect of Compound Rehmannia Granules on intestinal flora of the pneumonia model rats induced by Streptococcus pneumoniae(Spn)through 16s rRNA sequencing technology,and to clarify its potential mechanism.Methods:A total of 30 rats were randomly divided into control group(normal rats),model group(Spn-induced pneumonia rat models),low dose of Compound Rehmannia Granules group(pneumonia rat models administered 1.75 g·kg-1 Compound Rehmannia Granules by gavage),medium dose of Compound Rehmannia Granules group(pneumonia rat models administered 3.50 g·kg-1 Compound Rehmannia Granules by gavage),and high dose of Compound Rehmannia Granules group(pneumonia rat model administered 7.00 g·kg-1 Compound Rehmannia Granules by gavage),with 6 rats in each group.The wet/dry weight(W/D)ratio of lung tissue and blood gas indexes were measured;HE staining was used to observe the pathomorphology and the degree of lung injury in lung tissue of the rats in various groups were assessed;kit assay was used to detect the bacterial load level and the levels of interleukin(IL)-6,IL-8,and IL-10 in bronchoalveolar lavage fluid(BALF)of the rats in various groups;16s rRNA intestinal flora sequencing analysis was performed.Results:Compared with control group,the arterial partial pressure of carbon dioxide(PaCO2)of the rats in model group was significantly increased(P<0.05),and the arterial partial pressure of oxygen(PaO2)and oxygen saturation(SaO2)were significantly decreased(P<0.05);compared with model group,the PaCO2 of the rats in low,medium,and high doses of Compound Rehmannia Granules groups was significantly decreased(P<0.05),and the PaO? and SaO?were significantly increased(P<0.05),showing a dose-dependent manner.The HE staining results showed no significant injury in lung tissue of the rats in control group;the cells in lung tissue of the rats in model group were arranged disorderly with massive inflammatory cell infiltration,and the alveolar wall capillaries were significantly dilated;compared with model group,the morphological damage of lung tissue of the rats in low,medium,and high doses of Compound Rehmannia Granules groups was improved.Compared with control group,the W/D value and pathological score of lung tissue of the rats in model group were significantly increased(P<0.05);compared with model group,the W/D value and pathological scores of lung tissue of the rats in low,medium,and high doses of Compound Rehmannia Granules groups were significantly decreased(P<0.05),showing a dose-dependent manner.Compared with control group,the bacterial load level in BALF of the rats in model group was significantly increased(P<0.05);compared with model group,the bacterial load level in BALF of the rats in low,medium,and high doses of Compound Rehmannia Granules groups was significantly decreased(P<0.05),showing a dose-dependent manner.Compared with control group,the levels of IL-6 and IL-8 in BALF of the rats in model group were significantly increased(P<0.05),and the IL-10 level was significantly decreased(P<0.05);compared with model group,the levels of IL-6 and IL-8 in BALF of the rats in low,medium,and high doses of Compound Rehmannia Granules groups were significantly decreased(P<0.05),and the IL-10 level was significantly increased(P<0.05),showing a dose-dependent manner.Compared with control group,the microbial abundance indicator(Chao1),microbial diversity and evenness indicator(Shannon),microbial dominance indicator(Simpson),and observed species indicator(observed_species)of the rats in model group were significantly decreased(P<0.05);compared with model group,the Chao1,Shannon,Simpson,and(observed_species)indices of the rats in low,medium,and high doses of Compound Rehmannia Granules groups were significantly increased(P<0.05),showing a dose-dependent manner.Compared with control group,the relative abundance of Bacteroidetes in model group was decreased,the relative abundance of Firmicutes was increased,and the Firmicutes/Bacteroidetes ratio was significantly increased(P<0.05);compared with model group,the relative abundances of Bacteroidetes in low,medium,and high doses of Compound Rehmannia Granules groups were increased,the relative abundance of Firmicutes was decreased,and the Firmicutes/Bacteroidetes ratio was significantly decreased(P<0.05),showing a dose-dependent manner.At the family level,compared with control group,the relative abundances of Corynebacteriaceae,Staphylococcaceae,and Moraxellaceae in model group were significantly increased,while the relative abundances of Lactobacillaceae,Lachnospiraceae,and Akkermansiaceae were significantly decreased;compared with model group,the relative abundances of Corynebacteriaceae,Staphylococcaceae,and Moraxellaceae in low,medium,and high doses of Compound Rehmannia Granules groups were significantly decreased,and the relative abundances of Lactobacillaceae,Lachnospiraceae,and Akkermansiaceae were significantly increased.At the genus level,compared with control group,the relative abundances of Desulfovibrio and Facklamia in model group were significantly increased,and the relative abundances of Bifidobacterium and Ruminococcaceae were significantly decreased;compared with model group,the relative abundances of Desulfovibrio and Facklamia in low,medium,and high doses of Compound Rehmannia Granules groups were significantly decreased,and the relative abundances of Bifidobacterium and Ruminococcaceae were significantly increased.Conclusion:Compound Rehmannia Granules can alleviate inflammation and lung injury in Spn-induced pneumonia rats,which may be related to the increase in the abundance and diversity of intestinal flora.

Objective:To investigate the relationship between oxygen consumption (VO 2), carbon dioxide production (VCO 2), and Oxygen Consumption/lactate (VO 2/Lac) with risk of death in patients with severe ARDS. Methods:A retrospective cohort study method was used, and the study subjects were hospitalized for >5 days adult patients with severe ARDS in the central intensive care unit of Henan Provincial People's Hospital from 1 March 2020 to 30 June 2023. The following patients were excluded: IC test was not completed on the 4th day of ICU admission, IC test results were unreliable, mechanical ventilation duration had exceeded 48 h at the time of ICU transfer or admission, palliative care patients and pregnant and parturient women. Using indirect calorimetry to determine VO 2 and VCO 2 values on the 4th day of admission, reviewing medical records to obtain general condition, disease information, blood gas analysis (including lactate value), diagnostic and therapeutic measures, and following up deaths by telephone and time of death. The primary outcome measure was death at 90 days, and the secondary outcome measure was death at 28 days, length of stay in ICU, total length of stay, and total hospitalization cost. Cox regression analysis and linear regression analysis were used to investigate the relationship between VO 2, VCO 2, VO 2/Lac and primary and secondary outcome indexes. Results:A total of 216 patients were enrolled, 78 patients (36.1%) died and 138 patients (63.9%) survived at 90 days. After correction for confounders, the results of multifactorial Cox regression analysis suggested that compared with the Q4 group, HR (95% CI) for 90-day risk of death in the VO 2 Q1 and Q2 groups was 3.21 (1.38, 7.49) and 3.24 (1.42, 7.38), and HR (95% CI) for 90-day risk of death in the VCO 2 Q1, Q2 and Q3 groups was 5.88 (2.33, 14.84), 4.26 (1. 60, 11.34) and 3.54 (1.34, 9.35), respectively, and the HR (95% CI) for 90-day risk of death in the VO 2/Lac Q1, Q2 and Q3 groups were 8.72 (3.01, 25.25), 8.43 (2.91, 24.47) and 4.04 (1.34, 12.17) respectively. P-trends were all <0.05, indicating that VO 2, VCO 2 and VO 2/Lac were linearly and negatively associated with the risk of 90-day mortality. In addition, VO 2, VCO 2, and VO 2/Lac were negatively associated with 28-day risk of death and higher VO 2/Lac was negatively associated with length of ICU stay. Conclusions:VO 2, VCO 2 and VO 2/Lac were negatively associated with 90-day mortality risk and 28-day mortality risk in patients with severe ARDS and may be independent risk factors predicting mortality risk of such patients.

Objective To cultivate glioblastoma U87 stem-like cells(SLCs)and to detect the level of stemness bio-markers,mitochondrial respiratory capacity and the capacity of in vivo tumorigenesis.Methods B-27,growth factors EGF and bFGF was added into DMEM/F-12 culture in serum-free stem cell culture medium for U87 SLCs.Suspended culture of U87 SLCs was suspended using the neuro-sphere formation assay,while adherent culture of U87 SLCs was achieved by coating Matrigel matrix on the culture surface.The mRNA and protein level of stemness biomarkers in culture were detected using real-time quantitative PCR and Western blot.The proportion of CD133+cells in culture was detected by flow cytometry.The changes of cell oxygen consumption rate were detected by Seahorse cell metabo-lism analysis.Cell tumorigenesis ability was verified by subcutaneous tumor transplantation in animals.Results U87 SLCs in stem cell culture medium would grow into typical sphere morphology within one week,and the spheres would continue to grow as the culture process prolongs.At the appropriate concentration of adhesive,U87 SLCs adhered to and grow well in stem cell culture medium.The mRNA transcription of stemness biomarkers such as CD133,nes-tin,OLIG2,CD44,CD15,and integrin α6(ITGA6)was significantly increased as found in both culture methods,and the protein levels of CD133 and nestin were also increased under both methods(P＜0.05).U87 SLCs showed higher mitochondrial reserve respiratory capacity(P＜0.05).U87 SLCs could form larger subcutaneous tumors with fewer inoculated cells(P＜0.05),and grew faster in vivo with stronger tumorigenic ability.Conclusions U87 SLCs have typical stemness characteristics and may function as tumor cell model with higher stemness properties.

Objective:To report a case of Kufor-Rakeb syndrome caused by novel ATP13A2 mutation, collect the cases related to ATP13A2 gene mutation published in recent years, summarize the clinical manifestations of the disease, and broaden the clinical diagnostic thinking. Methods:The clinical manifestations of a newly diagnosed patient with Kufor-Rakeb syndrome caused by ATP13A2 gene mutation admitted to Zhongda Hospital, Southeast University on November 26, 2021, were summarized. The related cases of ATP13A2 mutation published from January 2000 to December 2021 were searched through the PubMed and CNKI databases using the keywords "ATP13A2" and "Parkinson′s disease". The onset age, clinical symptoms, family history, genetic testing, and levodopa responsiveness results of the patients were collected. Results:The patient is a 52-year-old female with the main clinical symptoms of static tremor and bradykinesia. Physical examination showed a gear like increase in muscle tension in the right upper limb, involuntary shaking of the right hand and slow movement. She had good responsiveness to levodopa, and the magnetic resonance imaging and susceptibility weighted imaging of the head showed a lack of clear observation of bilateral black matter swallowtail sign. Whole exome sequencing showed that mutations c.3010A>G (p.S1004G) and c.1195+5G>A (splice) were found in the ATP13A2 gene, both of which were not reported. The c.3010A>G (p.S1004G) mutation originated from the mother, and the c.1195+5G>A (splice) mutation originated from the father. In the retrospective literature review, a total of 10 cases were collected, with onset ages ranging from 18 months to 24 years. Among them, 4/10 patients′ parents married close relatives, and the clinical manifestations were mainly motor symptoms of Parkinson′s disease. In addition, 5/10 patients had cognitive dysfunction, and 3/10 patients had mental symptoms. And demonstrations of most patients′ magnetic resonance imaging were normal in the early stage of the disease, and as the disease progressed, some patients′ imaging results showed specific changes, such as whole brain atrophy and changes in the corpus callosum. Meanwhile, 8/10 patients showed good responsiveness to levodopa. Conclusions:Kufor-Rakeb syndrome is a special type of adolescent levodopa responsive Parkinson′s disease caused by ATP13A2 mutation, which is an autosomal recessive disorder. In addition to motor symptoms such as static tremor and bradykinesia, its clinical manifestations may also be accompanied by non motor symptoms such as cognitive and psychiatric disorders. The disease responds well to treatment with levodopa.