Lung cancer ranks the top of malignancies that cause cancer-related deaths worldwide.The leaves of Morus alba L are traditional Chinese medicine widely applied in respiratory diseases.Our previous work has demonstrated the anti-lung cancer effect of secondary metabolites of mulberry leaf,but their mechanism of action has still not fully elucidated.We synthesized Moracin N(MAN)-Probe conjugated with alkyne to label lung cancer cells and identified protein targets by chemical proteomic analysis.MAN and its probe exerted similar growth-inhibitory effect on human lung cancer cells.Chemical proteomic results showed that MAN targeted the programmed death ligand 1(PD-L1)checkpoint pathway and T cell receptor(TCR)signaling pathway,indicating its immune-regulatory function.Cell-free surface plasmon resonance(SPR)results showed the direct interaction of MAN with PD-L1 protein.Molecular docking analysis demonstrated that MAN bound to E158 residue of PD-L1 protein.MAN downregulated the expression levels of PD-L1 in a time-and dose-dependent manner and disrupted the PD-L1/programmed death 1(PD-1)binding,including other secondary metabolites of mulberry leaves Guangsangon E(GSE)and Chalcomoracin(CMR).Human peripheral blood mononuclear cells(PBMCs)co-cultured with MAN-treated A549 cells,resulting in the increase of CD8+GZMB+T cells and the decrease of CD8+PD-1+T cells.It suggested that MAN exerts anti-cancer effect through blocking the PD-L1/PD-1 signaling.In vivo,MAN combined with anti-PD-1 antibody significantly inhibited lung cancer development and metastasis,indicating their synergistic effect.Taken together,secondary metabolites of mulberry leaves target the PD-L1/PD-1 signaling,enhance T cell-mediated immunity and inhibit the tumorigenesis of lung cancer.Their modulatory effect on tumor microenvironment makes them able to enhance the therapeutic efficacy of immune checkpoint inhibitors in lung cancer.

Parkin, an E3 ubiquitin ligase, plays a role in maintaining mitochondrial homeostasis through targeting damaged mitochondria for mitophagy. Accumulating evidence suggests that the acetylation modification of the key mitophagy machinery influences mitophagy level, but the underlying mechanism is poorly understood. Here, our study demonstrated that inhibition of histone deacetylase (HDAC) by treatment of HDACis activates mitophagy through mediating Parkin acetylation, leading to inhibition of cervical cancer cell proliferation. Bioinformatics analysis shows that Parkin expression is inversely correlated with HDAC2 expression in human cervical cancer, indicating the low acetylation level of Parkin. Using mass spectrometry, Parkin is identified to interact with two upstream molecules, acetylase acetyl-CoA acetyltransferase 1 (ACAT1) and deacetylase HDAC2. Under treatment of suberoylanilide hydroxamic acid (SAHA), Parkin is acetylated at lysine residues 129, 220 and 349, located in different domains of Parkin protein. In in vitro experiments, combined mutation of Parkin largely attenuate the interaction of Parkin with PTEN induced putative kinase 1 (PINK1) and the function of Parkin in mitophagy induction and tumor suppression. In tumor xenografts, the expression of mutant Parkin impairs the tumor suppressive effect of Parkin and decreases the anticancer activity of SAHA. Our results reveal an acetylation-dependent regulatory mechanism governing Parkin in mitophagy and cervical carcinogenesis, which offers a new mitophagy modulation strategy for cancer therapy.

The global coronavirus disease 2019 epidemic is still in a pandemic state. Aging population with underlying diseases is prone to become severe, and have a higher mortality. The treatment capacity of the critical care department directly determines the treatment success rate of critical illness. At present, there is still a certain gap between domestic and foreign countries in intensive care unit (ICU), which is not only in the allocation of medical staff, but also in the beds and settings. The current medical model cannot fully meet the needs of development. The experience and lessons of many major public health emergencies suggested that " dual track of peace and war" approach in discipline construction of critical care is the best medical model. Following the concept of "combination of peace and war", strengthening the discipline construction of critical care department in municipal and district designated hospitals, allocating reasonable standard ICU, step-down ICU and combat readiness ICU, establishing rapid response team, and strengthening regular training and scientific management may be the key measures to deal with the epidemic.

OBJECTIVE：To discuss the inhibitory effect of lanthanum chloride on the calcification of vascular smooth muscle cells（VSMCs）induced by high phosphorus and its mechanism. METHODS ：On the basis of screening the action concentration and time of lanthanum chloride by MTT method ，human VSMCs were divided into control group （1 mmol/L phosphorus solution ）， lanthanum chloride high concentration control group （1 mmol/L phosphorus solution+ 60 μmol/L lanthanum chloride），model group （3 mmol/L phosphorus solution ），sodium chloride group （3 mmol/L phosphorus solution+ 180 μmol/L sodium chloride），nuclear factor κB（NF-κB）signaling pathway agonist+lanthanum chloride group （3 mmol/L phosphorus solution+ 1 μg/mL lipopolysaccharide+ 60 μmol/L lanthanum chloride），positive control group （3 mmol/L phosphorus solution+ 100 μmol/L sodium pyrophosphate），and lanthanum chloride low ，medium，and high concentration groups （3 mmol/L phosphorus solution+ 15，30，60 μmol/L lanthanum chloride）. Alizarin red S staining and Von Kossa staining were used to detect cell calcification in each group after treated with phosphorus solution for 6 d and relevant medicine for 2 d. Western blot assay was used to detect the protein expression of TNF-α receptor associated protein 6（TRAF6），nuclear factor κB inhibitor protein α（IκBα），NF-κB p65，bone morphogenetic protein 2 （BMP-2），smooth muscle 22 α（SM22α）and Runt related transcription factor 2（Runx2）. Real-time fluorescence quantitative polymerase chain reaction was used to detect mRNA expression of TRAF 6，IκBα，BMP-2，SM22α and Runx2. RESULTS ： Compared with control group ，no cell calcification was observed in the lanthanum chloride high concentration control group ，while obvious cell calcification and significant increase of OD value were observed in model group and sodium chloride group （P＜ 0.01）；protein and mRNA expression of TRAF 6 and BMP- 2 in cytoplasm as well as mRNA expression of Runx 2，protein expression of NF-κB p65 and Runx 2 in nucleus were significantly increased （P＜0.01）；protein and mRNA expression of IκBα and SM22α as well as protein expression of NF-κB p65 in cytoplasm were significantly decreased （P＜0.01）. Compared with model group，cell calcification was significantly improved in lanthanum chloride groups and positive control group ，while OD values were significantly reduced ；the expression levels of the above-mentioned protein and mRNA were reversed to varying degrees （P＜0.05 or P＜0.01）. Compared with lanthanum chloride high concentration group ，obvious cell calcification was observed in NF-κB signaling pathway agonist + lanthanum chloride group ，and OD value was significantly increased ；the above indexes were significantly reversed in cytoplasm and nucleus （P＜0.05 or P＜0.01）. CONCLUSIONS ：Lanthanum chloride can inhibit the calcification of VSMCs induced by high phosphorus ，and its mechanism may be related to the inhibition of NF-κB signaling pathway activation.

AIM:To investigate the effect of decitabine (Dacogen, DAC) on the proliferation and differentia-tion of K562 cells.METHODS:The K562 cells were treated with different concentrations of DAC .The colony formation ability of the cells was detected by the colony formation assay with semi-solid culture .The cell viability was detected with MTT assay.The morphologic features were observed under inverted microscope with Wright ’s staining.The changes of the cell cycle distribution and the expression of CD 11b and CD42b were analyzed with flow cytometry .The protein expression of CDK2, cyclin E1, P27, GATA-1 and PU.1 in the K562 cells was determined by Western blot .RESULTS:DAC signifi-cantly decreased the colony number of the cells and cell viability in a dose-dependent manner .The morphological changes of the cells displayed partial differentiation .After treated the K562 cells with DAC for 72 h, the cell proportion in S phase was obviously decreased , while the cell proportion in G 2/M phase was obviously increased in a dose-dependent manner . After treated the K562 cells with DAC for 7 d, the percentage of CD11b and CD14 positive cells was further elevated , and the protein expression of P27, GATA-1 and PU.1 was increased.However, the protein expression of CDK2 and cyclin E1 was decreased .CONCLUSION:DAC inhibits the proliferation and induces differentiation of the K 562 cells via regulation of cell cycle .

Objective To explore the clinical efficacy and safety of panaxadiol saponins for the treatment of non-small cell lung cancer(NSCLC) with chemotherapy-induced leukopenia.Methods 92 NSCLC patients with leukopenia after chemotherapy were selected and divided into the observation group (46 cases) and the control group (46 cases) by random number table method.On the second day after the chemotherapy,the observation group was given panaxadiol saponins capsules,3 tablets/time,2 times/day.The control group was orally given placebo or reserpine,4 weeks for one course of treatment,the two groups were continuously treated for two courses.The clinical efficacy,number of leukocytes,improvement of TCM symptoms and adverse reactions were evaluated.Results After treatment for 4 weeks and 8 weeks,the WBC counts of the observation group were (4.48 ±0.77) × 109/L and (4.92 ± 0.89) × 109/L,respectively,which were significantly higher than those of the control group[(4.02 ± O.93) × 109/L and (4.57 ± 0.86) × 109/L],the differences were statistically significant(t =8.24,8.41,all P ＜ 0.05).After treatment for 4 weeks and 8 weeks,the TCM syndrome scores of the observation group were (24.02 ± 5.91)points and (21.73 ± 4.14) points,respectively,which were lower than those of the control group [(26.33 ± 5.08) points and (23.14 ± 3.90) points],the differences were statistically significant (t =9.68,9.63,all P ＜ 0.05).After treatment for 4 weeks and 8 weeks,the total effective rates of TCM were 76.09％ (35/46) and 82.61％ (38/46),respectively,which were significantly higher than those of the control group [63.04％ (29/46) and 63.04％ (29/46)],the differences were statistically significant(x2 =10.32,8.61,all P ＜ 0.05).The effective rates of leukopenia improvement of the observation group after treatment for 4 weeks and 8 weeks were 69.57％ (32/46) and 78.26％ (36/46),respectively,which were higher than those of the control group [56.52％ (26/46) and 65.22％ (30/46)],the differences were statistically significant(t =9.38,9.51,all P ＜ 0.05).There were no significant differences in adverse reactions between the two groups (P ＞ 0.05).Conclusion Panaxadiol saponins in the treatment of NSCLC chemotherapy-induced leukopenia can significantly improve the number of white blood cells,improve the clinical symptoms,and it has good safety.

Aim To investigate the effect of bufalin on proliferation and expression of WT1 in K562 cells. Methods The colony number of K562 cell was detec-ted with semi-solid culture assay. The cell cycle was measured by flowcytometry, and the expression of WT1 was observed with immunocytochemistry. Subcutaneous tumor models established by K562 cells in BALB/C nu/nu mice were divided into three groups, including model group, bufalin group and positive control group. After 21 days, the subcutaneous tumors were removed for calculating the inhibitory rate of tumor growth. HE staining and immunohistochemistry were used to ob-serve the morphological changes and the expression of WT1 . Results ① Bufalin could significantly decrease the colony number of K562 cell, arrest it at G0/G1 phase and down-regulate its expression of WT1 in a dose-dependent manner. ② Compared with the model group, the tumor inhibitory rate was much higher, while the volume and the weight were obviously lower in the other two groups. ③Bufalin could induce apop-tosis, necrosis, hemorrhage and fibrosis with HE stai-ning, and down-regulate the expression of WT1. Con-clusion Bufalin could inhibit the proliferation, arrest the cell cycle at G0/G1 phase and down-regulate the expression of WT1 in vitro. Bufalin could inhibit the tumor inhibitory rate, the volume and the weight of the subcutaneous tumors, induce apoptosis, necrosis, hemorrhage and fibrosis with HE staining and down-regulate the expression of WT1 .

Cell Differentiation ; Cell Polarity ; Ginsenosides ; Humans ; K562 Cells ; Leukemia

Aim To observe mesenchymal stem cell differentiation into osteoblast induced by TSPG, and explore how TSPG enhances the promotion of hemato-poiesis of osteoblast differentiation from mesenchymal stem cell. Methods MSCs were cultured by TSPG combined with osteoinductive medium. The cellular vi-ability of proliferation was detected with MTT assay. The content of alkaline phosphatase in the cultural su-pernatant was tested with pNPP assay. The ability of MSCs to form calcium nodes was also observed after a-lizarin red stain. The protein expression of RUNX2 was analyzed with Western blot. The content of cytokines associated with hematopoiesis was tested with Elisa as-say. The ability of promoting hematopoiesis was detec-ted with hematopoietic colony forming assay. Results Both MTT and pNPP assay showed that optical den-sity ( OD) values were increased in response to TSPG treatment in a dose-dependent manner. The mineraliza-tion formation ability was enhanced with TSPG-treat-ment. Meanwhile, the expression of RUNX2 protein was up-regulated in TSPG-treated cell. Moreover, the content of cytokines associated with hematopoiesis and the number of hematopoietic progenitor colony were in-creased by TSPG-treatment compared with the control group. Conclusion TSPG could induce MSCs differ-entiation in to osteoblast and enhance the effect of oste-oblast differentiation from MSCs on promoting hemato-poiesis.

[ ABSTRACT] AIM:To observe the effects of total saponins of Panax ginseng ( TSPG) on the promotion of hema-topoiesis and to explore the underlying mechanism in treating aplastic anemia ( AA) in a mouse model.METHODS:For preparation of AA model, BALB/c mice were exposed to sublethal dose of 5.0 Gyγradiation, followed by transplantation of lymphocytes from DBA/2 donor mice.The experiment was divided into 6 groups, including normal control group, AA model group, TSPG treatment groups with low, medium and high doses, and positive control group with cyclosporine A ( CsA) .Both TSPG and CsA were administered by gastrogavage.After 15 d treatment, the peripheral hemogram was test-ed, the cytokine contents in serum were measured, and bone marrow semisolid culture of colony-forming assay was conduc-ted for determining hematopoietic progenitor cells.The protein levels of extracellular signal-regulated kinase 1/2 ( ERK1/2) and its phosphorylation status in the bone marrow cells were determined.RESULTS:Curative effect of TSPG in trea-ting AA mice was satisfactory.Peripheral counts of white blood cells and platelet, and concentration of hemoglobin in TSPG groups with medium and high doses were significantly higher than those in model control.TSPG increased the colony num-bers of CFU-E, BFU-E, CFU-GM and CFU-MK derived from hematopoietic progenitor cells.Meanwhile, up-regulation of the phosphorylated ERK1/2, decreased contents of Th1 cytokines and increased contents of Th2 cytokines in the serum were observed.CONCLUSION:TSPG possess the dual efficacies on the promotion of hematopoiesis and immunoregulation in AA mice by regulating the expression of Th1/Th2/Th17 cytokines and increasing the phosphorylation of ERK1/2.