Osteoporosis(OP) is a senile bone disease characterized by an imbalance between bone remodeling and bone formation. Targeting pathogenesis of kidney deficiency, spleen deficiency, and blood stasis, Bushen Jianpi Huoxue Decoction has a significant effect on the treatment of OP by tonifying kidney, invigorating spleen, and activating blood circulation. MicroRNA(miRNA) and the anti-apoptotic protein B-cell lymphoma-2-like protein 1(BCL2L1) are closely related to bone cell metabolism. Therefore, in this study, the binding of miR-140-5p to BCL2L1 was detected by dual luciferase assay and polymerase chain reaction(PCR). After silencing or overexpressing miR-140-5p, the apoptosis, autophagy, and osteogenic function of human fetal osteoblast cell line 1.19(HFOB1.19) were observed by flow cytometry and Western blot. Bushen Jianpi Huoxue Decoction-containing serum was prepared by intragastric administration of Bushen Jianpi Huoxue Decoction in rats. Different concentrations of Bushen Jianpi Huoxue Decoction-containing serum were used to treat HFOB1.19 with or without miR-140-5p mimic. The expression of osteogenic proteins in each group was observed, and the role of miR-140-5p/BCL2L1 in apoptosis and autophagy of HFOB1.19 was studied, along with the effect of Bushen Jianpi Huoxue Decoction on these processes. As indicated by the dual luciferase assay, miR-140-5p bound to BCL2L1. Flow cytometry and Western blot showed that miR-140-5p promoted apoptosis and inhibited autophagy in HFOB1.19. After intervention with high, medium, and low doses of Bushen Jianpi Huoxue Decoction-medicated serum, compared with the miR-140-5p NC group, the expression of osteocalcin(OCN), osteopontin(OPN), Runt-related transcription factor 2(RUNX2), and transforming growth factor beta 1(TGF-β1) decreased in the miR-140-5p mimic group, while the expression of bone morphogenetic protein 2(BMP2) showed no significant difference under high-dose intervention. Therefore, miR-140-5p/BCL2L1 can promote apoptosis and inhibit autophagy in HFOB1.19. Bushen Jianpi Huoxue Decoction can affect the osteogenic effect of miR-140-5p through BMP2.
MicroRNAs/metabolism* ; Autophagy/drug effects* ; Apoptosis/drug effects* ; Humans ; Drugs, Chinese Herbal/administration & dosage* ; Animals ; Cell Line ; bcl-X Protein/metabolism* ; Osteoblasts/metabolism* ; Rats ; Osteoporosis/physiopathology* ; Male ; Rats, Sprague-Dawley ; Osteogenesis/drug effects*

This paper aims to study the effect of Ziziphi Spinosae Semen extracts on chronic unpredictable mild stress(CUMS)-induced depression-like and insomnia behavior models of mice. The CUMS-induced depression-like and insomnia behavior model of mice was established by CUMS treatment for three weeks. The mice were randomly divided into control group, model group, positive drug diazepam group(2 mg·kg~(-1)), as well as low-dose group(1.95 g·kg~(-1)), medium-dose group(3.9 g·kg~(-1)), and high-dose group(7.8 g·kg~(-1)) of Ziziphi Spinosae Semen extracts, with 18 mice in each group. On the 15th day of modeling, the drug was administered intragastrically once a day for one week. Then, the pentobarbital sodium cooperative righting experiment, open field experiment, and elevated plus maze experiment were carried out, respectively. The contents of neurotransmitters 5-hydroxytryptamine(5-HT) and 5-hydroxyindoleacetic acid(5-HIAA) in serum and thalamus of mice, as well as the levels of corticotropin releasing hormone(CRH), adrenocorticotropic hormone(ACTH), and corticosterone(CORT) in serum, were determined by enzyme-linked immunosorbent assay(ELISA). The neuron damage in the hippocampus of mice was observed by hematoxylin-eosin(HE) staining and Nissl staining. Western blot was used to detect the expressions of tryptophan hydroxylase 2(TPH2), serotonin transporter(SERT), monoamine oxidase A(MAOA), five prime repressors under dual repression binding protein 1(Freud1), synaptic plasticity-related proteins [cellular gene FOS(C-FOS), postsynaptic density protein 95(PSD95), synapsin 1(SYN1), and activity-regulated cytoskeleton-associated gene(ARC)], blood-brain barrier(BBB) permeability-related proteins [zonula occludens 1(ZO-1), occludin, and claudin 1], inflammatory factors [NOD-, LRR-and pyrin domain-containing protein 3(NLRP3), apoptosis-associated speck-like protein(ASC), gasdermin D(GSDMD), caspase-3, and caspase-8], and antioxidant factors [nuclear factor erythroid 2-related factor 2(NRF2) and heme oxygenase 1(HO1)] in thalamic tissue of mice. The results indicated that compared with that in the model group, the sleep latency was significantly shortened, and the sleep duration was significantly prolonged in each dose group of Ziziphi Spinosae Semen extracts. The number of visits to the central area of the open field and the distance and time of visits were significantly increased in each dose group of Ziziphi Spinosae Semen extracts. In addition, the proportion of distance and time of entering the open arm area of the elevated plus maze was significantly increased in each dose group of Ziziphi Spinosae Semen extracts. The contents of 5-HT and 5-HIAA in serum and thalamus of mice increased to varying degrees in each dose group of Ziziphi Spinosae Semen extracts; the contents of CRH, ACTH, and CORT in serum of mice were significantly decreased. The protein expression of TPH2 was significantly increased. The protein expression of MAOA, SERT, and Freud1 was significantly decreased. Ziziphi Spinosae Semen extracts could also significantly reduce the protein expression of C-FOS but significantly increase the protein expression of PSD95, ARC, and SYN1. They could reduce the pathological damage of the hippocampus in mice and significantly increase the protein expression of ZO-1, occluding, and claudin 1. The protein expression of NLRP3, GSDMD, ASC, caspase-3, and caspase-8 in the thalamic tissue of mice was significantly decreased, and the protein expression of HO1 and NRF2 was significantly increased. In conclusion, Ziziphi Spinosae Semen extracts could effectively improve sleep disorders and depression-like behaviors in CUMS-induced model mice, which may be related to regulating the 5-HT anabolism process and hypothalamic-pituitary-adrenal(HPA) axis-related hormone levels, reducing pathological damage in the hippocampus, improving synaptic plasticity, repairing BBB integrity, and alleviating inflammatory response and oxidative stress damage.
Animals ; Ziziphus/chemistry* ; Mice ; Male ; Depression/psychology* ; Drugs, Chinese Herbal/administration & dosage* ; Sleep Initiation and Maintenance Disorders/psychology* ; Stress, Psychological/complications* ; Behavior, Animal/drug effects* ; Humans ; Disease Models, Animal

Cryopreservation is the primary technique for in vitro preservation of allogeneic tissue. However, its success is often hindered by factors such as low temperature, ischemia, and hypoxia. This study investigated the potential of Sini Decoction, known for its antioxidant and anti-apoptotic properties, to reduce cryopreservation-induced injury in rats' sciatic nerves. Sini Decoction was prepared according to the Chinese Pharmacopoeia, and its cytotoxicity on Rsc96 cells was assessed by using the CCK-8 method. Sini Decoction at concentrations of 4, 8, and 16 mg·mL~(-1), termed as low-(SL), medium-(SM), and high-(SH) doses group, was used for cryopreservation of rats' sciatic nerves. A normal control(NC) group and a fresh nerve control(fresh) group were set. Flow cytometry and TUNEL staining were used to detect the apoptosis of neural tissue cells after cryopreservation. Western blot was used to detect the expression of apoptosis-related proteins(Bcl-2, Bax, caspase-3, and caspase-8) and nerve regeneration proteins(NGF and BDNF) in vitro after cryopreservation. Oxidative damage of neural tissue after cryopreservation was evaluated by measuring levels of GSH, SOD, MDA, ROS, and ATP. Cryopreserved nerves were then used for allogeneic transplantation. One week after transplantation, CD4~+ and CD8~+ fluorescent double staining assessed inflammatory cell invasion in the transplanted nerve segment, and ELISA evaluated the expression of serum inflammatory factors(IL-1, IFN-γ, and TNF-α) in recipients. Twenty weeks after transplantation, electrophysiology and NF200 neurofilament staining were used to evaluate nerve regeneration. RESULTS:: showed that Sini Decoction at concentrations of below 32 mg·mL~(-1) exhibited no cytotoxicity to Rsc96 cells. During in vitro nerve cryopreservation, Sini Decoction significantly reduced cell apoptosis, ROS, and MDA production compared to the NC group. In the SH group, the protein expression of NGF and BDNF in vitro, as well as ATP, SOD, and GSH production, were significantly increased. In the rejection reaction one week after transplantation, compared to the fresh nerve transplantation group, the SL and SM groups showed reduced CD4~+ and CD8~+ T cell invasion in the transplanted nerve segment and down-regulated IL-1, IFN-γ, and TNF-α expression in recipient serum. Twenty weeks after transplantation, the electrophysiological test results of CMAP, NCV, and NF200 neurofilament protein fluorescent staining in the SM and SH groups were superior to those in the NC and fresh groups. These findings indicate that Sini Decoction offers protective benefits in the cryopreservation of rats' sciatic nerves and holds significant potential for the in vitro preservation of tissue and organs.
Animals ; Apoptosis/drug effects* ; Rats ; Oxidative Stress/drug effects* ; Sciatic Nerve/cytology* ; Cryopreservation ; Drugs, Chinese Herbal/administration & dosage* ; Male ; Rats, Sprague-Dawley ; Protective Agents/pharmacology*

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*

To investigate the effects of phosphorus fertilizer on the morphological traits, active ingredients and rhizosphere soil microbial community of Polygala tenuifolia. The phosphorus fertilizer was calculated in terms of P_2O_5. Five treatments were set up: 0(CK), 17(P1), 34(P2), 51(P3), and 68(P4) kg per Mu(1 Mu≈667 m~2). A randomized block design was adopted. Samples of P. tenuifolia and its rhizosphere soil were collected under different superphosphate fertilizer treatments. Illumina high-throughput sequencing was used to analyze the rhizosphere soil microbial community, 9 morphological traits were measured and the content of 11 active ingredients were determined. The results showed that the whole plant weight, shoot fresh weight, root weight, and root peel thickness were the highest under P1 treatment, increasing by 34.41%, 38.80%, 39.21%, and 3.17% respectively compared to CK. Under P2 treatment, the plant height, stem diameter, root thickness, and core thickness were significantly higher than CK. Phosphorus fertilizer had a significant impact on the content of tenuifolin, sibiricose A5, sibiricose A6, arillanin A, 3,6'-disinapoyl sucrose, and polygalaxanthone Ⅲ. Correlation analysis results showed that the relative abundance of Arthrobacter, Bacillus, norank＿f＿Vicinamibacteraceae, norank＿o＿Vicinamibacterales, MND1 and other bacteria, as well as the relative abundance of Neocosmospora, Paraphoma and other fungi were positively correlated with root diameter, wood core diameter, the whole plant weight, root weight, shoot fresh weight of P. tenuifolia. Bacillus, Neocosmospora, Subulicystidium were significantly positively correlated with oligosaccharides such as 3,6'-disinapoyl sucrose, sibiricose A5、sibiricose A6、glomeratose A、arillanin A and tenuifoliside C. Arthrobacter, Humicola, Aspergillus, Paraphoma were positively correlated with tenuifolin and norank＿f＿Vicinamibacteraceae, norank＿o＿Vicinamibacterales, Fusarium were positively correlated with polygalaxanthone Ⅲ. Evidently, appropriate phosphorus application is conducive to the growth and quality improvement of P. tenuifolia, and can increase the abundance of beneficial microorganisms in the soil.
Rhizosphere ; Phosphorus/pharmacology* ; Soil Microbiology ; Polygala/anatomy & histology* ; Fertilizers/analysis* ; Bacteria/metabolism* ; Soil/chemistry* ; Microbiota/drug effects* ; Plant Roots/metabolism*

In recent years, there have been frequent adverse reactions/events associated with traditional Chinese medicine(TCM), especially liver injury related to traditional non-toxic TCM, which requires adequate attention. Liver injury related to traditional non-toxic TCM is characterized by its sporadic and insidious nature and is influenced by various factors, making its detection and identification challenging. There is an urgent need to develop a strategy and method for early detection and recognition of traditional non-toxic TCM-related liver injury. This study was based on national adverse drug reaction monitoring center big data, integrating methodologies such as reporting odds ratio(ROR), network toxicology, and computational chemistry, so as to systematically research the risk signal identification and evaluation methods for TCM-related liver injury. The optimized ROR method was used to discover potential TCM with a risk of liver injury, and network toxicology and computational chemistry were used to identify potentially high-risk TCM. Additionally, typical clinical cases were analyzed for confirmation. An integrated strategy of "discovery via big data, identification via dry/wet method, confirmation via typical cases, and precise risk prevention and control" was developed to identify the risk of TCM-related liver injury. Corydalis Rhizoma was identified as a TCM with high risk, and its toxicity-related substances and potential toxicity mechanisms were analyzed. The results revealed that liver injury is associated with components such as tetrahydropalmatine and tetrahydroberberine, with potential mechanisms related to immune-inflammatory pathways such as the tumor necrosis factor signaling pathway, interleukin-17 signaling pathway, and Th17 cell differentiation. This paper innovatively integrated real-world evidence and computational toxicology methods, offering insights and technical support for establishing a risk discovery and identification strategy for TCM-related liver injury based on real-world big data, providing innovative ideas and strategies for guiding the safe and rational use of medication in clinical practices.
Corydalis/adverse effects* ; Drugs, Chinese Herbal/adverse effects* ; Humans ; Chemical and Drug Induced Liver Injury/etiology* ; Medicine, Chinese Traditional/adverse effects* ; Rhizome/adverse effects* ; Male ; Female

OBJECTIVES: To investigate the genomic characteristics and prognostic factors of juvenile myelomonocytic leukemia (JMML) with RAS mutations. METHODS: A retrospective analysis was conducted on the clinical data of JMML children with RAS mutations treated at the Hematology Hospital of Chinese Academy of Medical Sciences, from January 2008 to November 2022. RESULTS: A total of 34 children were included, with 17 cases (50%) having isolated NRAS mutations, 9 cases (27%) having isolated KRAS mutations, and 8 cases (24%) having compound mutations. Compared to children with isolated NRAS mutations, those with NRAS compound mutations showed statistically significant differences in age at onset, platelet count, and fetal hemoglobin proportion (P<0.05). Cox proportional hazards regression model analysis revealed that hematopoietic stem cell transplantation (HSCT) and hepatomegaly (≥2 cm below the costal margin) were factors affecting the survival rate of JMML children with RAS mutations (P<0.05); hepatomegaly was a factor affecting survival in the non-HSCT group (P<0.05). CONCLUSIONS Children with NRAS compound mutations have a later onset age compared to those with isolated NRAS mutations. At initial diagnosis, children with NRAS compound mutations have poorer peripheral platelet and fetal hemoglobin levels than those with isolated NRAS mutations. Liver size at initial diagnosis is related to the prognosis of JMML children with RAS mutations. HSCT can improve the prognosis of JMML children with RAS mutations.
Humans ; Leukemia, Myelomonocytic, Juvenile/therapy* ; Mutation ; Male ; Female ; Child, Preschool ; Retrospective Studies ; Child ; Infant ; GTP Phosphohydrolases/genetics* ; Membrane Proteins/genetics* ; Adolescent ; Hematopoietic Stem Cell Transplantation ; Proportional Hazards Models ; Proto-Oncogene Proteins p21(ras)/genetics* ; Prognosis

OBJECTIVES: To evaluate the application value of genetic newborn screening (gNBS) in the Yunnan region. METHODS: A prospective study was conducted with a random selection of 3 001 newborns born in the Yunnan region from February to December 2021. Traditional newborn screening (tNBS) was used to test biochemical indicators, and targeted next-generation sequencing was employed to screen 159 genes related to 156 diseases. Positive-screened newborns underwent validation and confirmation tests, and confirmed cases received standardized treatment and long-term follow-up. RESULTS: Among the 3 001 newborns, 166 (5.53%) were initially positive for genetic screening, and 1 435 (47.82%) were genetic carriers. The top ten genes with the highest variation frequency were GJB2 (21.29%), DUOX2 (7.27%), HBA (6.14%), GALC (3.63%), SLC12A3 (3.33%), HBB (3.03%), G6PD (2.94%), SLC25A13 (2.90%), PAH (2.73%), and UNC13D (2.68%). Among the initially positive newborns from tNBS and gNBS, 33 (1.10%) and 47 (1.57%) cases were confirmed, respectively. A total of 48 (1.60%) cases were confirmed using gNBS+tNBS. The receiver operating characteristic curve analysis demonstrated that the areas under the curve for tNBS, gNBS, and gNBS+tNBS in diagnosing diseases were 0.866, 0.982, and 0.968, respectively (P<0.05). DeLong's test showed that the area under the curve for gNBS and gNBS+tNBS was higher than that for tNBS (P<0.05). CONCLUSIONS gNBS can expand the range of disease detection, and its combined use with tNBS can significantly shorten diagnosis time, enabling early intervention and treatment.
Humans ; Infant, Newborn ; Neonatal Screening ; Genetic Testing ; Female ; Male ; Follow-Up Studies ; Prospective Studies ; China