This paper aimed to explore the effects of Duzhong Decoction(DZD)-containing serum on the proliferation and osteoblast differentiation of MC3T3-E1 cells through L-type voltage-gated calcium channels(L-VGCCs). L-VGCCs inhibitors, nifedipine and verapamil, were used to block L-VGCCs in osteoblasts. MC3T3-E1 cells were divided into a control group, a low-dose DZD-containing serum(L-DZD) group, a medium-dose DZD-containing serum(M-DZD) group, a high-dose DZD-containing serum(H-DZD) group, a nifedipine group, a H-DZD + nifedipine group, verapamil group, and a H-DZD + verapamil group. The CCK-8 method was used for cell proliferation analysis, alkaline phosphatase(ALP) assay kits for intracellular ALP activity measurement, Western blot for protein expression level in cells, real-time fluorescence quantitative PCR technology for intracellular mRNA expression level determination, fluorescence spectrophotometer for free Ca~(2+) concentration determination in osteoblasts, and alizarin red staining(ARS) for mineralized nodule formation in osteoblasts. The experimental results show that compared to the control group, DZD groups can promote MC3T3-E1 cell proliferation, ALP activity, and mineralized nodule formation, increase intracellular Ca~(2+) concentrations, and upregulate the protein expression of bone morphogenetic protein 2(BMP2), collagen Ⅰ(COL1), α2 subunit protein of L-VGCCs(L-VGCCα2), and the mRNA expression of Runt-related transcription factor 2(RUNX2), and BMP2. After blocking L-VGCCs with nifedipine and verapamil, the intervention effects of DZD-containing serum were inhibited to varying degrees. Both nifedipine and verapamil could inhibit ALP activity, reduce mineralized nodule areas, and downregulate the expression of bone formation-related proteins. Moreover, the effects of DZD-containing serum on increasing MC3T3-E1 cell proliferation, osteoblast differentiation, and Ca~(2+) concentrations, upregulating the mRNA expression of osteoprotegerin(OPG) and protein expression of phosphorylated protein kinase B(p-Akt) and phosphorylated forkhead box protein O1(p-FOXO1), and upregulating phosphatase and tensin homolog(PTEN) expression were reversed by nifedipine. The results indicate that DZD-containing serum can increase the Ca~(2+) concentration in MC3T3-E1 cells to promote bone formation, which may be mediated by L-VGCCs and the PTEN/Akt/FoxO1 signaling pathway, providing a new perspective on the mechanism of DZD in treating osteoporosis.
Animals ; Osteoblasts/metabolism* ; Cell Proliferation/drug effects* ; Cell Differentiation/drug effects* ; Mice ; Drugs, Chinese Herbal/pharmacology* ; Calcium Channels, L-Type/genetics* ; Alkaline Phosphatase/genetics* ; Serum/chemistry* ; Cell Line ; Osteogenesis/drug effects* ; Bone Morphogenetic Protein 2/genetics*

Objective To investigate the effect of E2 signaling in myofibers on muscular macrophage efferocytosis in mice with cardiotoxin-induced acute skeletal muscle injury.Methods Female wild-type C57BL/6 mice with and without ovariectomy and male C57BL/6 mice were given a CTX injection into the anterior tibial muscle to induce acute muscle injury,followed by intramuscular injection of β-estradiol(E2)or 4-hydroxytamoxifen(4-OHT).The changes in serum E2 of the mice were detected using ELISA,and the number,phenotypes,and efferocytosis of the macrophages in the inflammatory exudates and myofiber regeneration and repair were evaluated using immunofluorescence staining and flow cytometry.C2C12 cells were induced to differentiate into mature myotubes,which were treated with IFN-γ for 24 before treatment with β-Estradiol or 4-OHT.The treated myotubes were co-cultured with mouse peritoneal macrophages in a 1:2 ratio,followed by addition of PKH67-labeled apoptotic mouse mononuclear spleen cells induced by UV irradiation,and macrophage efferocytosis was observed using immunofluorescence staining and flow cytometry.Results Compared with the control mice,the female mice with ovariectomy showed significantly increased mononuclear macrophages in the inflammatory exudates,with increased M1 cell percentage,reduced M2 cell percentage and macrophage efferocytosis in the injured muscle,and obviously delayed myofiber regeneration and repair.In the cell co-culture systems,treatment of the myotubes with β-estradiol significantly increased the number and proportion of M2 macrophages and macrophage efferocytosis,while 4-OHT treatment resulted in the opposite changes.Conclusion In injured mouse skeletal muscles,myofiber E2 signaling promotes M1 to M2 transition to increase macrophage efferocytosis,thereby relieving inflammation and promoting muscle regeneration and repair.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates gene expression in a range of cells, including immune and epithelial cells. AhR signaling plays important roles in the immune system in both health and disease states. Tapinarof is a first-in-class small-molecule topical therapeutic AhR modulating agent launched for the treatment of psoriasis. To improve the activity and chemical stability of Tapinarof, a series of 2-phenylchromen-4-one derivatives were designed, synthesized and evaluated as novel AhR agonists. Compounds 5a, 5c, 5e and 5f potently activated AhR with an EC₅₀ value of 7, 9, 6 and 6 nmol·L^-1, respectively, which are 10-14 fold more potent than Tapinarof. Compounds 5a and 5e exhibit comparable inhibitory effects on IFN-γ production as Tapinarof. Furthermore, compounds 5a-5f exhibited favorable photochemical stability compared to Tapinarof. The compounds may eventually serve as lead compounds for the development of new AhR agonists.

Objective To investigate the effect of E2 signaling in myofibers on muscular macrophage efferocytosis in mice with cardiotoxin-induced acute skeletal muscle injury.Methods Female wild-type C57BL/6 mice with and without ovariectomy and male C57BL/6 mice were given a CTX injection into the anterior tibial muscle to induce acute muscle injury,followed by intramuscular injection of β-estradiol(E2)or 4-hydroxytamoxifen(4-OHT).The changes in serum E2 of the mice were detected using ELISA,and the number,phenotypes,and efferocytosis of the macrophages in the inflammatory exudates and myofiber regeneration and repair were evaluated using immunofluorescence staining and flow cytometry.C2C12 cells were induced to differentiate into mature myotubes,which were treated with IFN-γ for 24 before treatment with β-Estradiol or 4-OHT.The treated myotubes were co-cultured with mouse peritoneal macrophages in a 1:2 ratio,followed by addition of PKH67-labeled apoptotic mouse mononuclear spleen cells induced by UV irradiation,and macrophage efferocytosis was observed using immunofluorescence staining and flow cytometry.Results Compared with the control mice,the female mice with ovariectomy showed significantly increased mononuclear macrophages in the inflammatory exudates,with increased M1 cell percentage,reduced M2 cell percentage and macrophage efferocytosis in the injured muscle,and obviously delayed myofiber regeneration and repair.In the cell co-culture systems,treatment of the myotubes with β-estradiol significantly increased the number and proportion of M2 macrophages and macrophage efferocytosis,while 4-OHT treatment resulted in the opposite changes.Conclusion In injured mouse skeletal muscles,myofiber E2 signaling promotes M1 to M2 transition to increase macrophage efferocytosis,thereby relieving inflammation and promoting muscle regeneration and repair.

Objective:To investigate the effects of human umbilical cord mesenchymal stem cells exosomes (hucMSC-Exo) on depression-like behavior and polarization dynamic transition of hippocampal microglia in chronic unpredictable mild stress(CUMS) mice.Methods:The hucMSC was isolated, cultured, and the 5th generation hucMSC-Exo was extracted by ultracentrifugation and identified.Biological markers of CD9 and CD63 of hucMSC-Exo were detected by Western blot.A total of 12 SPF grade male C57BL/6J mice were randomly selected to establish depression model by CUMS method at the same time, and relevant behavioral experiments, including opening field test (OFT) and forced swimming test (FST) were used to detect changes of depression-like behavior in CUMS mice. After modeling, 12 mice were randomly divided into two groups, with 6 mice in each group. One group of mice was the exosome treatment group (CUMS+ Exo group) after stereotactic brain injection of hucMSC-Exo in the hippocampus, and the other group was the CUMS group. The expression of inflammatory signals in the hippocampus of mice in both groups were detected by micro PET/CT scanning technique. The proportion of M2-type microglia and M1-type microglia in the hippocampus of mice was detected by tissue immunofluorescence. β3-tubulin immunofluorescence staining was used to detect the length changes of neuronal axons in the depressed cell model constructed with corticosterone (CORT). EdU staining was used to detect neuronal proliferation. TUNEL staining was used to observe the apoptosis of neurons in the hippocampus of mice.Statistical analysis was conducted by GraphPad 8.0 software, and t-test was used for inter group comparison. Results:Under the electron microscope, hucMSC-Exo showed typical " double-layer cup-like" small vesicle-like changes and the particle diameter was about 100 nm.Western blot confirmed the expression of lconic proteins of CD9 and CD63. In the micro PET/CT scans, the uptake of [18F]DPA-714 in the CUMS+ Exo group was lower than that of the CUMS group, and the difference was statistically significant ((0.91±0.02)g/mL, (0.81±0.05)g/mL, t=4.54, P=0.001 1). In the opening field test, the percentage of central path length ((3.40±0.44)%, (5.17±0.90)%, t=4.33, P=0.001 5) and the time spent on the central path ((7.04±0.60)s, (10.22±1.41)s, t=6.02, P=0.000 1) in CUMS+ Exo group were higher than those in CUMS group, while the total distances of the two groups were not statistically significant ( t=0.48, P>0.05). In the forced swimming test, the immobility time in the CUMS+ Exo group was less than that in the CUMS group ((152.33±7.28) s, (133.50±4.32) s, t=5.45, P=0.000 3). In tissue immunofluorescence experiments, compared with the CUMS group, the proportion of M2-type microglia of hippocampus in the CUMS+ Exo group((0.33±0.04), (0.59±0.12), t=5.11, P=0.000 5)increased and the proportion of M1-type microglia ((0.56±0.06), (0.41±0.03), t=5.15, P=0.000 4) decreased in the CUMS+ Exo group. β3-tubulin-labeled immunofluorescence results showed an increase of neuronal axon length in the CORT+ Exo group compared with that in the CORT group((3.99±0.99) μm, (6.76±1.11) μm, t=6.10, P=0.000 1). The results of cell proliferation test showed an increase of proliferation rate in the CORT+ Exo group compared with that in the CORT group((0.74±0.07), (0.64±0.03), t=3.32, P=0.001 8). In the TUNEL staining experiment, the apoptosis rate of neurons in the hippocampal region of mice in the CUMS+ Exo group was lower than that of CUMS group ((0.24±0.04), (0.39±0.04), t=6.11, P=0.000 1). Conclusion:hucMSC-Exo can promote the conversion of M1 polarized microglia to M2 type microglia to alleviate depression-like behavior and reduce neuronal apoptosis in CUMS mice.

Objective:To investigate the predictive value of different body obesity measures for non-alcoholic fatty liver disease (NAFLD).Methods:It was a cross-sectional study. The present study was a case-control study involving 553 subjects who underwent physical examination from January to April 2022. The subjects were divided into NAFLD group ( n=321 cases) and control group ( n=232 cases) according to abdominal ultrasound imaging parameters. All subjects completed a general information questionnaire, liver ultrasound examination, serum biochemical indices and physical measurements. Logistic regression model was used to analyze the correlation between human obesity measures (neck circumference, triceps skinfold thickness (TSF),body mass index (BMI), waist-to-hip ratio, lipid accumulation index (LAP), visceral fat index (VAI), body roundness index (BRI) and a body shape index (ABSI)) and NAFLD. Receiver operating characteristic (ROC) curve and area under the curve (AUC) were used to evaluate the predictive value of single and combined measures of obesity for NAFLD. Results:The subjects were stratified by gender, and the quartile levels of BMI, neck circumference, TSF, waist-to-hip ratio, LAP, VAI and BRI were all correlated with NAFLD in both male and female (all P<0.05). After further adjustment for confounding factors, compared with those in group Q 1, group Q 4 of the above-mentioned indexes still had higher odds ratios ( P<0.05). The AUC value of LAP in predicting NAFLD was the largest in both men and women, which was 0.836(0.788-0.876) and 0.885(0.839-0.921), and the cut-off value was 41.93 and 33.27, respectively. There was no significant difference in AUC of ROC predicting NAFLD among LAP, BRI and BMI ( P>0.05). The AUC of ABSI in predicting NAFLD was less than 0.7(namely 0.584(0.525-0.641) and 0.679(0.618-0.735) in men and women, respectively), which indicated poor predictive performance for NAFLD. In the pairwise combination index, the AUC of ROC predicting NAFLD with TSF+LAP in male was the largest, which was 0.864(0.819-0.901), and there was statistical significance when compared with BRI (AUC=0.818(0.769-0.860)) and BMI (AUC=0.816(0.767-0.858)) ( P<0.05), but there was no statistical significance when compared with LAP (AUC=0.836(0.788-0.876)) ( P>0.05). The AUC of ROC predicting NAFLD with VAI+LAP in women was the largest, it was 0.894(0.849-0.928), there was statistical significance when compared with BMI (AUC=0.849(0.799-0.890)) ( P<0.05), but there was no statistical significance when compared with LAP (AUC=0.885(0.839-0.921)) and BRI (AUC=0.870(0.822-0.908)) ( P>0.05). Conclusion:BMI, neck circumference, TSF, waist-to-hip ratio, LAP, VAI and BRI all have good predictive value for NAFLD.

Acute Disease ; Aged ; Anti-Bacterial Agents/therapeutic use* ; Bacteremia/microbiology* ; Female ; Hepatitis B, Chronic/complications* ; Hepatitis C, Chronic/complications* ; Humans ; Hypersplenism/complications* ; Liver Cirrhosis/complications* ; Meningococcal Infections/microbiology* ; Neisseria meningitidis/isolation & purification* ; Peritonitis/microbiology*

OBJECTIVE: To explore the combined pro-apoptosis effect of HSP90 inhibitor BIIB021 and chloroquine (CQ) in chronic myeloid leukemia (CML) cells bearing T315I mutation and its mechanism. METHODS: The p210-T315I cells were divided into 4 groups by different treatment: control, BIIB021, CQ, and BIIB021 + CQ. After treated with BIIB021 or/and CQ for 24 hours, Annexin V/PI binding assay was used to detect apoptosis rates of CML cells. DAPI staining was used to observe nuclear fragmentation, and Western blot was used to detect the expression of caspase 3, PARP (apoptosis related proteins) and p62, LC3-I/II (autophagy related proteins). P210-T315I cells were inoculated subcutaneously into mice and CML mouse models were established. The mice in treatment groups were injected with BIIB021 and/or CQ while mice in control group were treated with PBS and normal saline. The tumor volume of mice was measured every 4 days, and protein level of cleaved-caspase 3 and LC3-II in tumor tissue were detected by immunohistochemistry. RESULTS: The results showed that BIIB021 induced apoptosis of CML cells in a dose-dependent manner ( r=0.91). CQ could enhance the apoptosis-inducing effect of BIIB021. Flow cytometry analysis results showed that the apoptosis rate of p210-T315I cells in combination group was higher than that in BIIB021 or CQ only group (P<0.05). DAPI staining showed nuclear fragmentation in combination group could be observed more obviously. Western blot analysis showed that BIIB021 could induce LC3-I to convert to LC3-II and decrease p62 protein levels (P<0.05). Moreover, the combination group had higher expression of LC3-II, p62 (P<0.05), activated PARP and activated caspase 3 than BIIB021 only group (P<0.05). Besides, experiment in vivo showed the mean tumor volume in co-treatment group was lower than that in single drug group (P<0.01). Immunohistochemistry of tumor tissue also showed the protein level of cleaved-caspase 3 and LC3-II in combined group was higher than that in BIIB021 only group. CONCLUSION HSP90 inhibitor BIIB021 induced significant apoptosis of CML cells bearing T315I both in vivo and in vitro. CQ can enhance this effect probably by autophagy inhibition.
Adenine/analogs & derivatives* ; Animals ; Apoptosis ; Autophagy ; Caspase 3/metabolism* ; Cell Line, Tumor ; Chloroquine/therapeutic use* ; Fusion Proteins, bcr-abl/pharmacology* ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy* ; Mice ; Mutation ; Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use* ; Pyridines

BACKGROUND: The significant morbidity and mortality resulted from the infection of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) call for urgent development of effective and safe vaccines. We report the immunogenicity and safety of an inactivated SARS-CoV-2 vaccine, KCONVAC, in healthy adults. METHODS: Phase 1 and phase 2 randomized, double-blind, and placebo-controlled trials of KCONVAC were conducted in healthy Chinese adults aged 18 to 59 years. The participants in the phase 1 trial were randomized to receive two doses, one each on Days 0 and 14, of either KCONVAC (5 or 10 μg/dose) or placebo. The participants in the phase 2 trial were randomized to receive either KCONVAC (at 5 or 10 μg/dose) or placebo on Days 0 and 14 (0/14 regimen) or Days 0 and 28 (0/28 regimen). In the phase 1 trial, the primary safety endpoint was the proportion of participants experiencing adverse reactions/events within 28 days following the administration of each dose. In the phase 2 trial, the primary immunogenicity endpoints were neutralization antibody seroconversion and titer and anti-receptor-binding domain immunoglobulin G seroconversion at 28 days after the second dose. RESULTS: In the phase 1 trial, 60 participants were enrolled and received at least one dose of 5-μg vaccine (n = 24), 10-μg vaccine (n = 24), or placebo (n = 12). In the phase 2 trial, 500 participants were enrolled and received at least one dose of 5-μg vaccine (n = 100 for 0/14 or 0/28 regimens), 10-μg vaccine (n = 100 for each regimen), or placebo (n = 50 for each regimen). In the phase 1 trial, 13 (54%), 11 (46%), and seven (7/12) participants reported at least one adverse event (AE) after receiving 5-, 10-μg vaccine, or placebo, respectively. In the phase 2 trial, 16 (16%), 19 (19%), and nine (18%) 0/14-regimen participants reported at least one AE after receiving 5-, 10-μg vaccine, or placebo, respectively. Similar AE incidences were observed in the three 0/28-regimen treatment groups. No AEs with an intensity of grade 3+ were reported, expect for one vaccine-unrelated serious AE (foot fracture) reported in the phase 1 trial. KCONVAC induced significant antibody responses; 0/28 regimen showed a higher immune responses than that did 0/14 regimen after receiving two vaccine doses. CONCLUSIONS: Both doses of KCONVAC are well tolerated and able to induce robust immune responses in healthy adults. These results support testing 5-μg vaccine in the 0/28 regimen in an upcoming phase 3 efficacy trial. TRIAL REGISTRATION http://www.chictr.org.cn/index.aspx (No. ChiCTR2000038804, http://www.chictr.org.cn/showproj.aspx?proj=62350; No. ChiCTR2000039462, http://www.chictr.org.cn/showproj.aspx?proj=63353).
Adult ; COVID-19 ; COVID-19 Vaccines ; Double-Blind Method ; Humans ; SARS-CoV-2 ; Vaccines, Inactivated/adverse effects*

Phyllanthi Fructus, a unique Chinese and Tibetan medicinal plant with both edible and medical values, has high potential of cultivation and development. The resources of Phyllanthi Fructus in China are rich, mainly distributed in Yunnan, Sichuan, Fujian, Guangdong, Guangxi, etc. Phyllanthi Fructus is widely used in the clinical practice of Chinese medicine and plays an important role in Tibetan medicine, Uyghur medicine, Yi medicine, and Mongolian medicine. Phyllanthi Fructus mainly contains phenolic acids,tannins, terpenes, sterols, fatty acids, flavonoids, amino acids and other compounds. Modern pharmacological studies show that Phyllanthi Fructus has antioxidant, anticancer, blood lipid-lowering, liver protective, antimicrobial, anti-inflammatory, and immune regulatory activities. In this paper, the research status of Phyllanthi Fructus was reviewed from the aspects of herbal textual research,chemical composition, and pharmacological action. The quality markers(Q-markers) of Phyllanthi Fructus were predicted and analyzed from the aspects of biogenic pathway, specificity and measurability of chemical components, efficacy, properties, new clinical uses, drug-food homology, and transformation of polyphenols. The results will provide a scientific basis for the quality control, quality evaluation, and standard formulation of Phyllanthi Fructus.
China ; Drugs, Chinese Herbal ; Fruit ; Medicine, Tibetan Traditional ; Quality Control