OBJECTIVE: To explore the effects of hydroxychloroquine (HCQ) on immune mediators dysregulation in severe infection after chemotherapy in acute myeloid leukemia (AML) and its molecular mechanism. METHODS: Bone marrow or peripheral blood samples of 36 AML patients with severe infection (AML-SI) and 29 AML patients without infection (AML-NI) after chemotherapy were collected from the First Affiliated Hospital of Gannan Medical University from August 2022 to June 2023. In addition, the peripheral blood of 21 healthy subjects from the same period in our hospital was selected as the control group. The mRNA expressions of CXCL12, CXCR4 and CXCR7 were detected by RT-qPCR technology, and the levels of IL-6, IL-8 and TNF-α were detected by ELISA. Leukemia-derived THP-1 cells were selected and constructed as AML disease model. At the same time, bone marrow mesenchymal stem cells (BM-MSCs) from AML-SI patients were co-cultured with THP-1 cells and divided into Mono group and Co-culture group. THP-1 cells were treated with different concentration gradients of HCQ. The cell proliferation activity was subsequently detected by CCK-8 method and apoptosis was detected by Annexin V/PI double staining flow cytometry. ELISA was used to detect the changes of IL-6, IL-8 and TNF-α levels in the supernatant of the cell co-culture system, RT-qPCR was used to detect the mRNA expression changes of the core members of the CXCL12-CXCR4/7 regulatory axis, and Western blot was used to detect the expressions of apoptosis regulatory molecules and related signaling pathway proteins. RESULTS: CXCL12, CXCR4, CXCR7, as well as IL-6, IL-8, and TNF-α were all abnormally increased in AML patients, and the increases were more significant in AML-SI patients (P <0.01). Furthermore, there were statistically significant differences between AML-NI patients and AML-SI patients (all P <0.05). HCQ could inhibit the proliferation and induce the apoptosis of THP-1 cells, but the low concentration of HCQ had no significant effect on the killing of THP-1 cells. When THP-1 cells were co-cultured with BM-MSCs of AML patients, the levels of IL-6, IL-8 and TNF-α in the supernatance of Co-culture group were significantly higher than those of Mono group (all P <0.01). After HCQ intervention, the levels of IL-6, IL-8 and TNF-α in cell culture supernatant of Mono group were significantly decreased compared with those before intervention (all P <0.01). Similarly, those of Co-culture group were also significantly decreased (all P <0.001). However, the expression of the core members of the CXCL12-CXCR4/7 regulatory axis was weakly affected by HCQ. HCQ could up-regulate the expression of pro-apoptotic protein Bax, down-regulate the expression of anti-apoptotic protein Bcl-2, as well as simultaneously promote the hydrolytic activation of Caspase-3 when inhibiting the activation level of TLR4/NF-κB pathway, then induce the programmed death of THP-1 cells after intervention. CONCLUSION The core members of CXCL12-CXCR4/7 axis and related cytokines may be important mediators of severe infectious immune disorders in AML patients. HCQ can inhibit cytokine levels to reverse immune mediators dysregulation and suppress malignant biological characteristics of leukemia cells. The mechanisms may be related to regulating the expression of Bcl-2 family proteins, hydrolytically activating Caspase-3 and inhibiting the activation of TLR4/NF-κB signaling pathway.
Humans ; Leukemia, Myeloid, Acute/immunology* ; Hydroxychloroquine/pharmacology* ; Receptors, CXCR4/metabolism* ; Apoptosis/drug effects* ; Tumor Necrosis Factor-alpha/metabolism* ; Chemokine CXCL12/metabolism* ; Interleukin-8/metabolism* ; Interleukin-6/metabolism* ; Receptors, CXCR/metabolism* ; Mesenchymal Stem Cells ; THP-1 Cells

In recent years, researchers, pharmaceutical companies, and political makers gradually using more real-world data (RWD) to produce real-world evidence (RWE) for policy-making. A research team of Harvard University launched the RCT DUPLICATE project in 2018, aiming to replicate 30 randomized controlled trials using the medical claims database in order to explore methods for quantifying the efficacy-effectiveness gap and explain its potential sources, to enhance the credibility of the RWE. This paper reviews the background of RCT DUPLICATE Initiative, highlights the research purposes, research design and implementation process of the RCT DUPLICATE Initiative, to help domestic scholars better understand the scope and application value of RWE.
Humans ; Randomized Controlled Trials as Topic ; Cognition ; Databases, Factual ; Research Personnel ; Universities

With the promotion and application of big medical data, non-interventional real-world evidence (RWE) has been used by regulators to assess the effectiveness of medical products. This paper briefly introduces the latest progress and research results of the RCT DUPLICATE Initiative launched by the research team of Harvard University in 2018 and summarizes relevant research experience based on the characteristics of China's medical service to provide inspiration and reference for domestic scholars to conduct related RWE research in the future.
Humans ; Randomized Controlled Trials as Topic ; Cognition ; Big Data ; Universities

OBJECTIVE: To develop a convenient method for rapid purification of fresh Pheretima proteins and assess the inhibitory effect of these proteins against pulmonary fibrosis. METHODS: The crude extract of fresh Pheretima was obtained by freeze-drying method and then purified by size exclusion chromatography. The composition of the purified proteins was analyzed by mass spectrometry. MRC-5 cells were treated with 5 ng/mL TGF-β1 alone (model group) or in combination with SB431542 (2 μmol/L) or the purified proteins (13.125 μg/mL), and the cytotoxicity of purified proteins and their inhibitory effects on cell proliferation were detected with CCK8 assay. Flow cytometry was used to detect the changes in cell apoptosis, and the cellular expressions of α-SMA, Vimentin, E-cadherin, collagen I, Smad2/3 and P-Smad2/3 were detected using RT-PCR and Western blotting. In the animal experiment, adult male C57BL/6 mice were subjected to intratracheal instillation of bleomycin followed by treatment with the purified proteins (5 mg/mL) for 21 days, after which HE and Masson staining was used to observe the pathological changes in the lung tissue of the mice. RESULTS: We successfully obtained purified proteins from fresh Pheretima protein by size exclusion chromatography. Treatment with the purified proteins significantly inhibited TGF-β1-induced proliferation of MRC-5 cells (P < 0.01), reduced the cellular expressions of α-SMA, Vimentin and collagen I (P < 0.001 or P < 0.01), increased the expression of E-cadherin (P < 0.01), and inhibited the expressions of Smad2/3 and P-Smad2/3 (P < 0.001 or P < 0.01). In male C57BL/6 mice models of bleomycin-induced pulmonary fibrosis, treatment with the purified proteins obviously reduced the number of inflammatory cells and fibrotic area in the lungs. CONCLUSION The purified proteins from fresh Pheretima obtained by size exclusion chromatography can inhibit pulmonary fibrosis in mice by regulating the TGF-β/ Smad pathway.
Animals ; Biological Products/pharmacology* ; Bleomycin/adverse effects* ; Cadherins/metabolism* ; Collagen Type I ; Lung/pathology* ; Male ; Mice ; Mice, Inbred C57BL ; Oligochaeta/chemistry* ; Pulmonary Fibrosis/drug therapy* ; Transforming Growth Factor beta1/metabolism* ; Vimentin/metabolism*

The Myh3 (myosin heavy chain 3) gene is a marker gene of muscle cell differentiation and regulates the utilization of energy in muscle cells, but whether it affects the glycolysis process of muscle cells in different states is rarely reported. In this paper, the expression patterns of Myh3 and glycolysis-related genes Pkm (M-type pyruvate kinse), Prkag3 (protein kinase adenosine monophosphate-activated γ3-subunit), and Gsk3β (glycogen synthase kinase-3β) were studied by the qRT-PCR (quantitative-Real-Time-PCR) method using C2C12 cells at different stages of myoblast and adipogenic differentiation as models. It was found that in the process of myoblast differentiation of C2C12 cells, the relative expression trends of Myh3 and glycolysis genes Prkag3 and Pkm were basically the same, and the relative expression levels first increased, reached the peak on the second day of differentiation, and then decreased; glycogen synthase the expression trend of the inhibitory gene Gsk3β was relatively stable. In the process of adipogenic differentiation of C2C12 cells, the relative expression trend of Myh3 and glycolysis genes Prkag3 and Pkm remained basically the same, and the relative expression gradually increased, reaching the highest value on the 8th day of differentiation; glycogen synthase inhibitory gene Gsk3β expression remained stable. In the myogenic differentiation state of C2C12 cells, qRT-PCR and Western blotting were used to detect the effects of interfering Myh3 on the mRNA and protein expressions of glycolysis-related genes Pkm, Prkag3, and Gsk3β. The results showed that after interfering with Myh3, the mRNA expressions of glycolysis genes Pkm and Prkag3 were significantly decreased (P<0.01), while the mRNA expression of glycogen synthase inhibitory gene Gsk3β had no significant change (P > 0.05). The protein levels of Myh3 and Pkm were significantly lower than those in the blank group and NC group. Under the adipogenic differentiation state of C2C12 cells, after interfering with Myh3, the mRNA levels of glycogen synthase inhibitor Gsk3β and glycolysis gene Prkag3 were significantly increased (P<0.01), and the mRNA level of glycolysis gene Pkm was decreased; the protein levels of Myh3 and Pkm in the Myh3 interference group were also lower than those in the blank group and NC group. Based on the above studies, there are significant differences in the levels of glycolysis in C2C12 cells in the myogenic and adipogenic states, and the expression patterns of Myh3 and glycolysis genes are similar. Further results showed that Myh3 suppression could inhibit the glycolysis of C2C12 cells in the myogenic state without affecting the glycogen synthesis. Unlike in the myogenic state, interfering expression of Myh3 in the adipogenic state of C2C12 cells inhibited both glycogen synthesis and glycolysis.

OBJECTIVE: To explore the clinical therapeutic effect of light and heat of moxibustion for knee osteoarthritis (KOA). METHODS: A total of 216 patients with KOA were randomized into a traditional moxibustion group (72 cases, 8 cases dropped off), a moxibustion light group (72 cases, 9 cases dropped off) and a moxibustion heat group (72 cases, 10 cases dropped off).The special light-heat separation moxibustion cup was applied, the patients in the traditional moxibustion group received the treatment of moxibustion, the patients in the moxibustion light group received the treatment of moxibustion light and the patients in the moxibustion heat group received the treatment of moxibustion heat. The acupoint selection of the three groups was Neixiyan (EX-LE 4), Dubi (ST 35) and Zusanli (ST 36), the treatment was given 20 min each time, 3 times a week, 4 weeks were required totally, and the follow-up surveys were made 4 and 8 weeks after treatment. The scores of Western Ontario and McMaster University osteoarthritis index (WOMAC) and visual analogue scale (VAS) were observed before treatment, after 2 and 4 weeks of treatment, 4 and 8 weeks after treatment. The therapeutic effects were evaluated according to the criterion of patient global assessment (PGA) after 4 weeks of treatment and 8 weeks after treatment. RESULTS: Compared with before treatment, the pain scores, stiffness scores, physical function scores and total scores of WOMAC were reduced after 2, 4 weeks of treatment and 4 weeks after treatment in the three groups (<0.05). The pain scores, stiffness scores, physical function scores and total scores of WOMAC were reduced 8 weeks after treatment in the traditional moxibustion group and the moxibustion heat group (<0.05). The stiffness score, physical function score and total score of WOMAC were reduced 8 weeks after treatment in the moxibustion light group (<0.05). The pain score, physical function score and total score of WOMAC in the traditional moxibustion group after 4 weeks of treatment were lower than the moxibustion light group (<0.05). Compared with before treatment, the VAS scores were reduced after 2, 4 weeks of treatment and 4 , 8 weeks after treatment in the three groups (<0.05). The improvement rates in the traditional moxibustion group and the moxibustion heat group after 4 weeks of treatment and 8 weeks after treatment were superior to the moxibustion light group (<0.05). CONCLUSION The light and heat of moxibustion have therapeutic effect for KOA, and the therapeutic effect of moxibustion heat is superior to moxibustion light.
Acupuncture Points ; Hot Temperature ; Humans ; Light ; Moxibustion ; Osteoarthritis, Knee ; therapy ; Treatment Outcome

The purpose of this research is to study the anti-atherosclerotic effects and mechanisms of berberine (BBR) in high fat diet (HFD) fed ApoE^-/- mice, and then to lay a solid foundation of the clinical studies of BBR treatment. The hyperlipidemic ApoE^-/- mice model was established by feeding HFD for 12 weeks. Mice were randomly divided into control group (chow diet), model group, BBR group (BBR-L: 50 mg·kg^-1, BBR-H: 150 mg·kg^-1) and atorvastatin (5 mg·kg^-1) group. Mice were intragastric administration with BBR in 0.5% sodium salt of caboxy methyl cellulose. After 12 weeks, enface aortas were stained with oil red O, and the lesions area were analyzed by Image J software. The inflammatory factor levels were detected by suspension microarray kits. Liver total cholesterol (TC), triglyceride (TG) and free fatty acids (FFA) were determined by commercial kits. Western blot was performed to examine the inflammatory pathway related and cholesterol and lipid transport related proteins' expression. All animal experiments were performed in accordance with the Regulation on the Administration of Laboratory Animals of Institute of Medicinal Biotechnology. After 12 weeks treatment, compared with model group, BBR treatment significantly reduced the lesions area of en face aortas and obviously inhibited serum proinflammatory factors such as IL-1β and IL-6 compared with model group. In addition, BBR treatment obviously reduced liver TC, TG and FFA levels compared with model group. Furthermore, mechanic study showed that BBR significantly inhibited MAPKs and NF-κB pathways, and increased cholesterol and lipid regulated proteins expression such as p-AMPK, LDLR, ABCA1 and SR-BI. In conclusion, BBR can obviously reduce enface aortas lesions in ApoE^-/- mice, which is related to inhibit inflammation and liver cholesterol and lipid accumulation.

Objective: To explore the expression and prognostic significance of miR-223 in patients with mantle cell lymphoma (MCL) and to investigate the possible mechanism. Methods: Twenty-one newly diagnosed MCL patients with bone marrow involvement were enrolled in the present study, 20 healthy donors as normal control. The expression level of miR-223 and SOX11 mRNA was determined by RQ-PCR. CCK-8 and flow cytometer assays were used to analyze cell proliferation, cell cycle and apoptosis of the constructed miR-223 overexpressing MCL cell line, Granta519 cells. SOX11 protein expression level was determined by Western blot. The target gene of miR-223 was confirmed by dual luciferase reporter assay. Results: ①Of the 21 newly diagnosed MCL patients, 15 were male and 6 female, the median age was 58 (37-72) years. The expression level of miR-223 was significantly down regulated in MCL patients compared with that of healthy donors (14.7±10.5 vs 1 244.1±1 935.2, P<0.001). The lower expression of miR-223 was inversely correlated with high-risk mantle international prognostic index (P=0.001), elevated LDH (P=0.001), ECOG score ≥2 (P=0.035). ②Using the median relative expression level of miR-223 as the cutoff value, 21 MCL patients were divided into high-expression group (n=10) and low-expression group (n=11) and found that the high-expression group had a significantly superior OS (median OS: 36 vs 12 months, P=0.021). ③In vitro results showed that compared with the control group, the proliferation of miR-223 overexpressed Granta519 cells was inhibited (the most significant reduction on 96h, P<0.001), manifested by lower proportion of cells in G2/M phase (P<0.001) and increased apoptosis (P<0.001), and the expression level of SOX11 protein in Granta519 cells was significantly lower than that of the control group. ④miR-223 could inhibited the 3' untranslated region of SOX11, and the expression level of miR-223 was significantly negatively correlated with mRNA level of SOX11 in MCL patients (r=-0.81, P<0.001). Conclusions: The expression of miR-223 was repressed in MCL and was associated with poor clinical outcomes, which may be probably attributed to its direct targeting SOX11.
Adult ; Aged ; Female ; Humans ; Lymphoma, Mantle-Cell ; Male ; MicroRNAs ; Middle Aged ; Prognosis ; RNA, Messenger ; SOXC Transcription Factors

Chinese medicinal formulae are the important means of clinical treatment in traditional Chinese medicine. It is urgent to use modern advanced scientific and technological means to reveal the complicated mechanism of Chinese medicinal formulae because they have the function characteristics of multiple components, multiple targets and integrated regulation. The systematic and comprehensive research model of proteomic is in line with the function characteristics of Chinese medicinal formulae, and proteomic has been widely used in the study of pharmacological mechanism of Chinese medicinal formulae. The recent applications of proteomic in pharmacological study of Chinese medicinal formulae in anti-cardiovascular and cerebrovascular diseases, anti-liver disease, antidiabetic, anticancer, anti-rheumatoid arthritis and other diseases were reviewed in this paper, and then the future development direction of proteomic in pharmacological study of Chinese medicinal formulae was put forward. This review is to provide the ideas and method for proteomic research on function mechanism of Chinese medicinal formulae.

Total glucosides of peony (TGP), containing the effective components of paeoniflorin (Pae), albiflorin (Alb) and so on, are effective parts of Radix Paeoniae Alba. And it possesses extensive pharmacological actions, one of which is hepatoprotective effect. In recent years, abundant of pharmacokinetics and pharmacodynamics research of TGP in hepatoprotective effects have been performed. However, the relative medicine of TGP in hepatoprotective effect has not been developed for clinical application. In order to provide reference for the development and rational clinical application of TGP, the research progresses of pharmacokinetics and pharmacodynamics of TGP in hepatoprotective effect were summarized in this paper. Pharmacokinetics research has clarified the process of absorption, distribution, metabolism and excretion of TGP in vivo, and liver injury disease can significantly influence its metabolic processes. Pharmacodynamics studies suggested that TGP can protect against acute liver injury, non-alcoholic fatty liver diseases (NAFLD), chronic liver fibrosis and liver cancer. However, the action mechanism and in vivo process about hepatoprotective effects of TGP have not been clearly revealed. How liver injury influences the metabolism of TGP and its integrated regulation through multiple targets need to be further studied. The combined pharmacokinetics and pharmacodynamics studies should be performed in favour of medicine development and clinical application of TGP in hepatoprotective effects.