BACKGROUND: T cell dysfunction, which includes exhaustion, anergy, and senescence, is a distinct T cell differentiation state that occurs after antigen exposure. Although T cell dysfunction has been a cornerstone of cancer immunotherapy, its potential in transplant research, while not yet as extensively explored, is attracting growing interest. Interferon regulatory factor 4 (IRF4) has been shown to play a pivotal role in inducing T cell dysfunction. METHODS: A novel ultra-low-dose combination of Trametinib and Rapamycin, targeting IRF4 inhibition, was employed to investigate T cell proliferation, apoptosis, cytokine secretion, expression of T-cell dysfunction-associated molecules, effects of mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) signaling pathways, and allograft survival in both in vitro and BALB/c to C57BL/6 mouse cardiac transplantation models. RESULTS: In vitro , blockade of IRF4 in T cells effectively inhibited T cell proliferation, increased apoptosis, and significantly upregulated the expression of programmed cell death protein 1 (PD-1), Helios, CD160, and cytotoxic T lymphocyte-associated antigen (CTLA-4), markers of T cell dysfunction. Furthermore, it suppressed the secretion of pro-inflammatory cytokines interferon (IFN)-γ and interleukin (IL)-17. Combining ultra-low-dose Trametinib (0.1 mg·kg -1 ·day -1 ) and Rapamycin (0.1 mg·kg -1 ·day -1 ) demonstrably extended graft survival, with 4 out of 5 mice exceeding 100 days post-transplantation. Moreover, analysis of grafts at day 7 confirmed sustained IFN regulatory factor 4 (IRF4) inhibition, enhanced PD-1 expression, and suppressed IFN-γ secretion, reinforcing the in vivo efficacy of this IRF4-targeting approach. The combination of Trametinib and Rapamycin synergistically inhibited the MAPK and mTOR signaling network, leading to a more pronounced suppression of IRF4 expression. CONCLUSIONS Targeting IRF4, a key regulator of T cell dysfunction, presents a promising avenue for inducing transplant immune tolerance. In this study, we demonstrate that a novel ultra-low-dose combination of Trametinib and Rapamycin synergistically suppresses the MAPK and mTOR signaling network, leading to profound IRF4 inhibition, promoting allograft acceptance, and offering a potential new therapeutic strategy for improved transplant outcomes. However, further research is necessary to elucidate the underlying pharmacological mechanisms and facilitate translation to clinical practice.
Animals ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Interferon Regulatory Factors/metabolism* ; Heart Transplantation/methods* ; T-Lymphocytes/immunology* ; Sirolimus/therapeutic use* ; Pyridones/therapeutic use* ; Graft Survival/drug effects* ; Pyrimidinones/therapeutic use* ; Cell Proliferation/drug effects* ; Apoptosis/drug effects* ; Male ; Signal Transduction/drug effects*

Ecological cultivation is an important way for the sustainable production of traditional Chinese medicine in the context of the carbon peaking and carbon neutrality goals. Facility cultivation and simulative habitat cultivation modes have been developed and applied to develop the endangered Dendrobium huoshanense on the basis of protection. However, the differences in the greenhouse gas emissions and global warming potential of these cultivation modes remain unexplored, which limits the accurate assessment of carbon-friendly ecological cultivation modes of D. huoshanense. Greenhouse gas emission flux monitoring based on the static chamber method provides an effective way to solve this problem. Therefore, this study conducted a field experiment in the facility cultivation and simulative habitat cultivation modes at a D. huoshanense cultivation base in Dabie Mountains, Anhui Province. From April 2023 to March 2024, samples of greenhouse gases were collected every month, and the concentrations of CO_2, CH_4, and N_2O of the samples were then detected by gas chromatography. The greenhouse gas emission fluxes, cumulative emissions, and global warming potential were further calculated, and the following results were obtained.(1)The two cultivation modes of D. huoshanense showed significant differences in greenhouse gas emission fluxes, especially the CO_2 emission flux, with a pattern of facility cultivation>simulative habitat cultivation [(35.60±11.70)mg·m~(-2)·h~(-1) vs(2.10±4.59)mg·m~(-2)·h~(-1)].(2) The annual cumulative CO_2 emission flux in the case of facility cultivation was significantly higher than that of simulative habitat cultivation[(3 077.00±842.00)kg·hm~(-2) vs(221.00±332.00)kg·hm~(-2)], while no significant difference was found in annual cumulative CH_4 and N_2O emission fluxes.(3) The facility cultivation mode had a significantly higher global warming potential than the simulative habitat cultivation mode [(3 053.00±847.00)kg·hm~(-2) vs(196.00±362.00)kg·hm~(-2)]. Overall, the simulative habitat cultivation of D. huoshanense has obvious carbon-friendly characteristics compared with facility cultivation, which is in line with the concept of ecological cultivation of medicinal plants. This study is of great reference significance for the implementation and promotion of the ecological cultivation mode of D. huoshanense under carbon peaking and carbon neutrality goals.
Dendrobium/chemistry* ; Greenhouse Gases/metabolism* ; Carbon/analysis* ; Ecosystem ; Carbon Dioxide/metabolism* ; China ; Global Warming

To improve the quality evaluation system of Hibisci Mutabilis Folium, this study established high performance liquid chromatography(HPLC) fingerprints of Hibisci Mutabilis Folium and evaluated the quality differences of medicinal materials from different places of production by chemometrics. Furthermore, a content measurement method of differential components was established based on quantitative analysis of multi-components by single-marker(QAMS). The fingerprints of 17 batches of Hibisci Mutabilis Folium from different places of production were constructed, with a total of 19 common peaks marked and seven components confirmed. The similarity between the sample fingerprints and the reference fingerprints ranged from 0.890 to 0.974. By utilizing principal component analysis(PCA), hierarchical cluster analysis(HCA), and orthogonal partial least squares-discriminant analysis(OPLS-DA), the chemical patterns of fingerprints were identified. Five components that could be used to evaluate the quality differences of Hibisci Mutabilis Folium were screened, namely peak 6(quercetin 3-O-β-robinobioside), peak 7(rutin), peak 9(kaempferol-3-O-β-robinobioside), peak 10(kaempferol-3-O-rutinoside), and peak 14(tiliroside). The relative correction factors of isoquercitrin, kaempferol-3-O-β-robinobioside, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside, and tiliroside were measured with rutin as the internal reference. The QAMS method was established for the content measurement of six flavonoids, and the results showed there was no significant difference compared to the results obtained by an external standard method. In summary, the HPLC fingerprints and QAMS method established in the study, demonstrating stability and accuracy, can provide a reference for the overall quality evaluation of Hibisci Mutabilis Folium.
Chromatography, High Pressure Liquid/methods* ; Drugs, Chinese Herbal/chemistry* ; Quality Control ; Principal Component Analysis

This study was aimed at developing an in vitro fluorescent substrate assay for the activity of leucyl aminopeptid-ase(LAP)from Echinococcus multilocularis and comparing it with the chemical chromogenic substrate enzyme activity assay.Through the establishment of reaction conditions for the fluorescent substrate-based in vitro enzyme activity assay,we com-pared the differences between the fluorescent substrate L-Leucine-7-amido-4-methylocoumarin(Leu-AMC)and the chemical chromogenic substrate L-Leucine-4-nitroanilide(Leu-pNA)through molecular docking,inhibition rates,and precision measures.Molecular docking revealed that the fluorescent substrate Leu-AMC had higher affinity for the protein than the chemical chromogenic substrate Leu-pNA.Through analysis of the effects of varying reaction conditions on fluorescence intensi-ty,we optimized the fluorescent substrate enzyme activity assay to demonstrate favorable performance at a reaction temperature of 37℃,a pH of 9.0,a protein concentration of 800 nmol/L,and a reaction duration of 60 minutes.Leu-AMC exhibited significant and distinct responses at a 5 μmol/L substrate concentration,under varying substrate conditions.The fluo-rescent substrate assay demonstrated more significant intergroup differences than the chemical chromogenic substrate assay when various inhibitors were added.This study established a fluorescence-based enzyme activity assay for leucyl aminopeptidase from Echinococcus multilocularis by using Leu-AMC as the substrate;this method demonstrated a more significant intergroup difference and sensitivity than the chemical chromogenic substrate assay.

Objective:To investigate the impact of minocycline on myocardial injury in diabetes cardiomyopathy(DCM)rats by regulating 5'-AMP activated protein kinase(AMPK)/sirtuin 1(SIRT1)/peroxisome proliferator activator receptor gamma coactiva-tor 1α(PGC-1α)signal pathway.Methods:SD rats were fed with high-fat diet for 4 weeks,and then a single intraperitoneal injection of 35 mg/kg streptozotocin was used to induce the DCM model.They were randomly grouped into model group,low-dose minocycline(20 mg/kg)group,high-dose minocycline(40 mg/kg)group,high-dose minocycline+Dorsomorphin(AMPK inhibitor,0.2 mg/kg)group,with 12 rats in each group,another 12 normal rats were fed with normal feed for 4 weeks,and then were given a single intraper-itoneal injection of the same dose of citric acid buffer,which was set as a sham operation group,after the intervention with minocy-cline and Dorsomorphin,and the fasting blood glucose(FBG),total cholesterol(TC)and triglyceride(TG)were measured;left ven-tricular ejection fraction(LVEF)and left ventricular short axis shortening(FS)were measured by ultrasound;HE staining and Mas-son staining were applied to detect the pathological morphology of myocardial tissue of rats in each group;the levels of serum and myo-cardial tissue inflammation IL-6,IL-18,and myocardial tissue oxidative stress indicators-superoxide dismutase(SOD)and malondial-dehyde(MDA)were measured with the kit;Western blot was applied to detect the expression of AMPK/SIRT1/PGC-1α pathway pro-tein in myocardial tissue of rats in each group.Results:Compared with the sham operation group,the myocardial tissue in the model group was seriously damaged,the levels of FBG,TC and TG,the cross-sectional area of myocardial cells and the proportion of myocar-dial fiber area,the levels of IL-6 and IL-18 in serum and myocardial tissue,and the level of MDA in myocardial tissue were obviously increased(P<0.05),the levels of LVEF,FS and SOD in myocardial tissue,and the protein expression of p-AMPK/AMPK,SIRT1 and PGC-1α were obviously decreased(P<0.05);compared with the model group,the myocardial tissue damage of rats in the low-dose and high-dose minocycline groups were reduced,the levels of FBG,TC and TG,the cross-sectional area of myocardial cells and the proportion of myocardial fiber area,the levels of IL-6 and IL-18 in serum and myocardial tissue,and the level of MDA in myocardial tissue were decreased(P<0.05),the LVEF,the levels of FS and SOD in myocardial tissue,and the protein expression of p-AMPK/AMPK,SIRT1 and PGC-1α were increased(P<0.05);compared with the low-dose minocycline group,the myocardial tissue damage in the high-dose minocycline group was further reduced,the levels of FBG,TC and TG,the cross-sectional area of myocardial cells and the proportion of myocardial fiber area,the levels of IL-6 and IL-18 in serum and myocardial tissue,and the level of MDA in myo-cardial tissue were further decreased(P<0.05),the levels of LVEF,FS and SOD in myocardial tissue,and the protein expression of p-AMPK/AMPK,SIRT1 and PGC-1α were further increased(P<0.05);compared with the high-dose minocycline group,the myocar-dial tissue damage of rats in the high-dose minocycline+Dorsomorphin group increased,the levels of FBG,TC and TG,the cross-sec-tional area of myocardial cells and the proportion of myocardial fiber area,the levels of IL-6 and IL-18 in serum and myocardial tis-sue,and the level of MDA in myocardial tissue were increased(P<0.05),the LVEF,the levels of FS and SOD in myocardial tissue,and the protein expression of p-AMPK/AMPK,SIRT1 and PGC-1α were decreased(P<0.05).Conclusion:Minocycline can reduce oxidative stress and inflammation in DCM rats by activating AMPK/SIRT1/PGC-1α signal,and improve glycolipid metabolism,thereby reducing myocardial injury and repairing cardiac function in rats.

This study was aimed at developing an in vitro fluorescent substrate assay for the activity of leucyl aminopeptid-ase(LAP)from Echinococcus multilocularis and comparing it with the chemical chromogenic substrate enzyme activity assay.Through the establishment of reaction conditions for the fluorescent substrate-based in vitro enzyme activity assay,we com-pared the differences between the fluorescent substrate L-Leucine-7-amido-4-methylocoumarin(Leu-AMC)and the chemical chromogenic substrate L-Leucine-4-nitroanilide(Leu-pNA)through molecular docking,inhibition rates,and precision measures.Molecular docking revealed that the fluorescent substrate Leu-AMC had higher affinity for the protein than the chemical chromogenic substrate Leu-pNA.Through analysis of the effects of varying reaction conditions on fluorescence intensi-ty,we optimized the fluorescent substrate enzyme activity assay to demonstrate favorable performance at a reaction temperature of 37℃,a pH of 9.0,a protein concentration of 800 nmol/L,and a reaction duration of 60 minutes.Leu-AMC exhibited significant and distinct responses at a 5 μmol/L substrate concentration,under varying substrate conditions.The fluo-rescent substrate assay demonstrated more significant intergroup differences than the chemical chromogenic substrate assay when various inhibitors were added.This study established a fluorescence-based enzyme activity assay for leucyl aminopeptidase from Echinococcus multilocularis by using Leu-AMC as the substrate;this method demonstrated a more significant intergroup difference and sensitivity than the chemical chromogenic substrate assay.

Objective:To investigate the impact of minocycline on myocardial injury in diabetes cardiomyopathy(DCM)rats by regulating 5'-AMP activated protein kinase(AMPK)/sirtuin 1(SIRT1)/peroxisome proliferator activator receptor gamma coactiva-tor 1α(PGC-1α)signal pathway.Methods:SD rats were fed with high-fat diet for 4 weeks,and then a single intraperitoneal injection of 35 mg/kg streptozotocin was used to induce the DCM model.They were randomly grouped into model group,low-dose minocycline(20 mg/kg)group,high-dose minocycline(40 mg/kg)group,high-dose minocycline+Dorsomorphin(AMPK inhibitor,0.2 mg/kg)group,with 12 rats in each group,another 12 normal rats were fed with normal feed for 4 weeks,and then were given a single intraper-itoneal injection of the same dose of citric acid buffer,which was set as a sham operation group,after the intervention with minocy-cline and Dorsomorphin,and the fasting blood glucose(FBG),total cholesterol(TC)and triglyceride(TG)were measured;left ven-tricular ejection fraction(LVEF)and left ventricular short axis shortening(FS)were measured by ultrasound;HE staining and Mas-son staining were applied to detect the pathological morphology of myocardial tissue of rats in each group;the levels of serum and myo-cardial tissue inflammation IL-6,IL-18,and myocardial tissue oxidative stress indicators-superoxide dismutase(SOD)and malondial-dehyde(MDA)were measured with the kit;Western blot was applied to detect the expression of AMPK/SIRT1/PGC-1α pathway pro-tein in myocardial tissue of rats in each group.Results:Compared with the sham operation group,the myocardial tissue in the model group was seriously damaged,the levels of FBG,TC and TG,the cross-sectional area of myocardial cells and the proportion of myocar-dial fiber area,the levels of IL-6 and IL-18 in serum and myocardial tissue,and the level of MDA in myocardial tissue were obviously increased(P<0.05),the levels of LVEF,FS and SOD in myocardial tissue,and the protein expression of p-AMPK/AMPK,SIRT1 and PGC-1α were obviously decreased(P<0.05);compared with the model group,the myocardial tissue damage of rats in the low-dose and high-dose minocycline groups were reduced,the levels of FBG,TC and TG,the cross-sectional area of myocardial cells and the proportion of myocardial fiber area,the levels of IL-6 and IL-18 in serum and myocardial tissue,and the level of MDA in myocardial tissue were decreased(P<0.05),the LVEF,the levels of FS and SOD in myocardial tissue,and the protein expression of p-AMPK/AMPK,SIRT1 and PGC-1α were increased(P<0.05);compared with the low-dose minocycline group,the myocardial tissue damage in the high-dose minocycline group was further reduced,the levels of FBG,TC and TG,the cross-sectional area of myocardial cells and the proportion of myocardial fiber area,the levels of IL-6 and IL-18 in serum and myocardial tissue,and the level of MDA in myo-cardial tissue were further decreased(P<0.05),the levels of LVEF,FS and SOD in myocardial tissue,and the protein expression of p-AMPK/AMPK,SIRT1 and PGC-1α were further increased(P<0.05);compared with the high-dose minocycline group,the myocar-dial tissue damage of rats in the high-dose minocycline+Dorsomorphin group increased,the levels of FBG,TC and TG,the cross-sec-tional area of myocardial cells and the proportion of myocardial fiber area,the levels of IL-6 and IL-18 in serum and myocardial tis-sue,and the level of MDA in myocardial tissue were increased(P<0.05),the LVEF,the levels of FS and SOD in myocardial tissue,and the protein expression of p-AMPK/AMPK,SIRT1 and PGC-1α were decreased(P<0.05).Conclusion:Minocycline can reduce oxidative stress and inflammation in DCM rats by activating AMPK/SIRT1/PGC-1α signal,and improve glycolipid metabolism,thereby reducing myocardial injury and repairing cardiac function in rats.

OBJECTIVE To learn the practical experience of medical insurance payment standards adjustment in Japan and South Korea， which will serve as a reference for the improvement of simple renewal mechanism in China. METHODS Retrieving relevant literature from CNKI and related policy documents from official websites of Japan and South Korea， the medical insurance payment standards adjustment practice in Japan and South Korea would be elucidated from 2 perspectives of adjustment criteria and formulas， and then were compared with the current simple renewal mechanism in China to clarify the areas where simple renewal mechanism in China can be optimized and propose several suggestions. RESULTS & CONCLUSIONS In terms of adjustment methods， Japan and South Korea were similar to China. For excessive drugs， the reduction rate of drugs was calculated based on the situation of excess and adjustments were implemented； however， there were differences in the specific adjustment criteria and formulas. Japan and South Korea adopted a linear price reduction approach for drugs with significant oversupply， while China adopted a gradient price reduction approach for drugs with both current and expected oversupply. The results of the comparative analysis show that China has initially established simple renewal mechanisms that are in line with the national conditions and the actual medical insurance situation， and has taken some innovative measures， including considering the current and expected oversupply of drugs and introducing a halving mechanism in the adjustment formula. However， there are also certain shortcomings， such as a relatively single set of indicators for adjusting conditions and a too broad range of gradient price reduction in adjustment formulas， which fail to fully reflect the market-oriented mechanism of “volume for price”. It is recommended that China’s medical insurance department increase consideration of drug fund expenditures， refine the gradient price reduction range of adjustment formulas， increase policy preferences for special category drugs when adding new indications， and further improve the mechanism for simple renewal.

Patients with severe trauma require an extremely timely treatment and transfusion plays an irreplaceable role in the emergency treatment of such patients. An increasing number of evidence-based medicinal evidences and clinical practices suggest that patients with severe traumatic bleeding benefit from early transfusion of low-titer group O whole blood or hemostatic resuscitation with red blood cells, plasma and platelet of a balanced ratio. However, the current domestic mode of blood supply cannot fully meet the requirements of timely and effective blood transfusion for emergency treatment of patients with severe trauma in clinical practice. In order to solve the key problems in blood supply and blood transfusion strategies for emergency treatment of severe trauma, Branch of Clinical Transfusion Medicine of Chinese Medical Association, Group for Trauma Emergency Care and Multiple Injuries of Trauma Branch of Chinese Medical Association, Young Scholar Group of Disaster Medicine Branch of Chinese Medical Association organized domestic experts of blood transfusion medicine and trauma treatment to jointly formulate Chinese expert consensus on blood support mode and blood transfusion strategies for emergency treatment of severe trauma patients ( version 2024). Based on the evidence-based medical evidence and Delphi method of expert consultation and voting, 10 recommendations were put forward from two aspects of blood support mode and transfusion strategies, aiming to provide a reference for transfusion resuscitation in the emergency treatment of severe trauma and further improve the success rate of treatment of patients with severe trauma.

Background::Homoharringtonine (HHT) is an effective anti-inflammatory, anti-viral, and anti-tumor protein synthesis inhibitor that has been applied clinically. Here, we explored the therapeutic effects of HHT in a mouse heart transplant model.Methods::Healthy C57BL/6 mice were used to observe the toxicity of HHT in the liver, kidney, and hematology. A mouse heart transplantation model was constructed, and the potential mechanism of HHT prolonging allograft survival was evaluated using Kaplan–Meier analysis, immunostaining, and bulk RNA sequencing analysis. The HHT-T cell crosstalk was modeled ex vivo to further verify the molecular mechanism of HHT-induced regulatory T cells (Tregs) differentiation. Results::HHT inhibited the activation and proliferation of T cells and promoted their apoptosis ex vivo. Treatment of 0.5 mg/kg HHT for 10 days significantly prolonged the mean graft survival time of the allografts from 7 days to 48 days ( P <0.001) without non-immune toxicity. The allografts had long-term survival after continuous HHT treatment for 28 days. HHT significantly reduced lymphocyte infiltration in the graft, and interferon-γ-secreting CD4 + and CD8 + T cells in the spleen ( P <0.01). HHT significantly increased the number of peripheral Tregs (about 20%, P <0.001) and serum interleukin (IL)-10 levels. HHT downregulated the expression of T cell receptor (TCR) signaling pathway-related genes ( CD4, H2-Eb1, TRAT1, and CD74) and upregulated the expression of IL-10 and transforming growth factor (TGF) -β pathway-related genes and Treg signature genes ( CTLA4, Foxp3, CD74, and ICOS). HHT increased CD4 + Foxp3 + cells and Foxp3 expression ex vivo, and it enhanced the inhibitory function of inducible Tregs. Conclusions::HHT promotes Treg cell differentiation and enhances Treg suppressive function by attenuating the TCR signaling pathway and upregulating the expression of Treg signature genes and IL-10 levels, thereby promoting mouse heart allograft acceptance. These findings may have therapeutic implications for organ transplant recipients, particularly those with viral infections and malignancies, which require a more suitable anti-rejection medication.