Osteoporosis is a prevalent skeletal condition characterized by reduced bone mass and strength, leading to increased fragility. Buqi-Tongluo (BQTL) decoction, a traditional Chinese medicine (TCM) prescription, has yet to be fully evaluated for its potential in treating bone diseases such as osteoporosis. To investigate the mechanism by which BQTL decoction inhibits osteoclast differentiation in vitro and validate these findings through in vivo experiments. We employed MTS assays to assess the potential proliferative or toxic effects of BQTL on bone marrow macrophages (BMMs) at various concentrations. TRAcP experiments were conducted to examine BQTL's impact on osteoclast differentiation. RT-PCR and Western blot analyses were utilized to evaluate the relative expression levels of osteoclast-specific genes and proteins under BQTL stimulation. Finally, in vivo experiments were performed using an osteoporosis model to further validate the in vitro findings. This study revealed that BQTL suppressed receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis and osteoclast resorption activity in vitro in a dose-dependent manner without observable cytotoxicity. The inhibitory effects of BQTL on osteoclast formation and function were attributed to the downregulation of NFATc1 and c-fos activity, primarily through attenuation of the MAPK, NF-κB, and Calcineurin signaling pathways. BQTL's inhibitory capacity was further examined in vivo using an ovariectomized (OVX) rat model, demonstrating a strong protective effect against bone loss. BQTL may serve as an effective therapeutic TCM for the treatment of postmenopausal osteoporosis and the alleviation of bone loss induced by estrogen deficiency and related conditions.
Animals ; NFATC Transcription Factors/genetics* ; Drugs, Chinese Herbal/pharmacology* ; Ovariectomy ; Osteoclasts/metabolism* ; Female ; Osteogenesis/drug effects* ; Rats, Sprague-Dawley ; Rats ; NF-kappa B/genetics* ; Osteoporosis/genetics* ; Signal Transduction/drug effects* ; Bone Resorption/genetics* ; Cell Differentiation/drug effects* ; Humans ; RANK Ligand/metabolism* ; Mitogen-Activated Protein Kinases/genetics* ; Transcription Factors

Panax ginseng(PG)and Panax notoginseng(PN)are highly valuable Chinese medicines(CM).Although both CMs have similar active constituents,their clinical applications are clearly different.Over the past decade,RNA sequencing(RNA-seq)analysis has been employed to investigate the molecular mechanisms of extracts or monomers.However,owing to the limited number of samples in standard RNA-seq,few studies have systematically compared the effects of PG and PN spanning multiple conditions at the transcriptomic level.Here,we developed an approach that simultaneously profiles transcriptome changes for multiplexed samples using RNA-seq(TCM-seq),a high-throughput,low-cost workflow to molecularly evaluate CM perturbations.A species-mixing experiment was conducted to illustrate the accuracy of sample multiplexing in TCM-seq.Transcriptomes from repeated samples were used to verify the robustness of TCM-seq.We then focused on the primary active components,Panax notoginseng sa-ponins(PNS)and Panax ginseng saponins(PGS)extracted from PN and PG,respectively.We also char-acterized the transcriptome changes of 10 cell lines,treated with four different doses of PNS and PGS,using TCM-seq to compare the differences in their perturbing effects on genes,functional pathways,gene modules,and molecular networks.The results of transcriptional data analysis showed that the tran-scriptional patterns of various cell lines were significantly distinct.PGS exhibited a stronger regulatory effect on genes involved in cardiovascular disease,whereas PNS resulted in a greater coagulation effect on vascular endothelial cells.This study proposes a paradigm to comprehensively explore the differences in mechanisms of action between CMs based on transcriptome readouts.

Objective:To identify the genome-wide characteristics and variations of the novel coronavirus 2019(2019-nCoV) by combining high-throughput sequencing and bioinformatics techniques, to type and trace the virus.Methods:Pharyngeal swab sample of one 2019-nCoV infection case imported from Weihai city was selected for 2019-nCoV whole-genome sequence capture and NGS high-throughput sequencing. The obtained sequencing data were sequenced and analyzed using virus mutation analysis software, we classify viruses and build phylogenetic trees to track their potential sources.Results:The complete genome sequence of 2019-nCoV was obtained, with a total length of 29 808 bp and an average sequencing depth of 5 013.11×. A total of 51 nucleotide mutations were found in 8 coding regions (ORF1ab, S, ORF3a, E, M, ORF6, ORF7a and N). The result of virus typing showed that the virus belonged to Omicron BA.2. Evolutionary analysis showed that the virus sequence was in the same clade as the reference strains from Japan.Conclusions:The sequencing method and analysis result established in this study can be used for mutation analysis of 2019-nCoV and case tracing, which is of great significance for the prevention and control of COVID-19.

Objective:To study the effects and mechanism of hyperbaric oxygen (HBO) on apoptosis and growth factor of skeletal muscle in rats with type 2 diabetes.Methods:A total of 24 GK rats with blood sugar >16.7 mmol/L, selected from 32 GK rats after 1 week feeding, were divided into model group ( n=8), metformin group ( n=8), and HBO group ( n=8), according to random number table method. The left 8 rats with blood sugar ≤ 16.7 mmol/L were included into control group. The rats in the control group and the model group were irrigated with pure water 5 ml·kg -1·d -1. The rats in the HBO group were given HBO every day on the basis of the treatment of the control group and the model group. After 3 weeks, the rats were executed. The tail blood was collected to measure fasting blood glucose. The right gastrocnemius muscle tissues were collected and made into paraffin sections for HE staining. The morphological and structural changes of gastrocnemius muscle in each group were observed under a light microscope. Immunohistochemistry was used to detect the expressions of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), cytokine, and Caspase-3; and the integrated optical density (IOD) was measured and calculated for comparison. TUNEL method was used to detect gastrocnemius cell apoptosis, and the apoptosis index was calculated. Results:After the rats were executed at 21 d, the 12 h fasting blood glucose of the rats in the model group, the metformin group, and the HBO group were all significantly higher than that of the control group [(7.26±0.78, 6.90±0.74, 6.92±0.95 vs. 4.74±0.53) mmol/L, P<0.05 in each comparison]. The expression of skeletal muscle bFGF in the rats of the control group was the highest, while that of the model group was the lowest. The expressions of skeletal muscle bFGF in the rats of the metformin group and the HBO group were obviously higher than that of the model group, but lower than that of the control group, with statistically significant difference ( P<0.05). The expressions of skeletal muscle VEGF in the rats of the metformin group and the HBO group were obviously higher than those of the model group and the control group, with statistically significant difference ( P<0.05). The expression of skeletal muscle eNOS in the rats of the control group was the highest, while those of the model group, the HBO group, and the metformin group were low. There were no statistical differences among the four groups ( P>0.05). The expressions of skeletal muscle Caspase-3 and the gastrocnemius cell apoptosis indexes of the metformin group and the HBO group were significantly lower than those of the model group, but higher than those of the control group, with statistical difference ( P<0.05) in all comparison. Conclusion:HBO can improve the skeletal muscle structure of the rats with diabetes, improve the expressions of bFGF and VEGF, decrease the expression of Caspase-3, and reduce cell apoptosis; it, therefore, has a certain protective effect on the skeletal muscle in rats with diabetes.

Objective:To study the effects and mechanism of hyperbaric oxygen (HBO) on apoptosis and growth factor of skeletal muscle in rats with type 2 diabetes.Methods:A total of 24 GK rats with blood sugar >16.7 mmol/L, selected from 32 GK rats after 1 week feeding, were divided into model group ( n=8), metformin group ( n=8), and HBO group ( n=8), according to random number table method. The left 8 rats with blood sugar ≤ 16.7 mmol/L were included into control group. The rats in the control group and the model group were irrigated with pure water 5 ml·kg -1·d -1. The rats in the HBO group were given HBO every day on the basis of the treatment of the control group and the model group. After 3 weeks, the rats were executed. The tail blood was collected to measure fasting blood glucose. The right gastrocnemius muscle tissues were collected and made into paraffin sections for HE staining. The morphological and structural changes of gastrocnemius muscle in each group were observed under a light microscope. Immunohistochemistry was used to detect the expressions of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), cytokine, and Caspase-3; and the integrated optical density (IOD) was measured and calculated for comparison. TUNEL method was used to detect gastrocnemius cell apoptosis, and the apoptosis index was calculated. Results:After the rats were executed at 21 d, the 12 h fasting blood glucose of the rats in the model group, the metformin group, and the HBO group were all significantly higher than that of the control group [(7.26±0.78, 6.90±0.74, 6.92±0.95 vs. 4.74±0.53) mmol/L, P<0.05 in each comparison]. The expression of skeletal muscle bFGF in the rats of the control group was the highest, while that of the model group was the lowest. The expressions of skeletal muscle bFGF in the rats of the metformin group and the HBO group were obviously higher than that of the model group, but lower than that of the control group, with statistically significant difference ( P<0.05). The expressions of skeletal muscle VEGF in the rats of the metformin group and the HBO group were obviously higher than those of the model group and the control group, with statistically significant difference ( P<0.05). The expression of skeletal muscle eNOS in the rats of the control group was the highest, while those of the model group, the HBO group, and the metformin group were low. There were no statistical differences among the four groups ( P>0.05). The expressions of skeletal muscle Caspase-3 and the gastrocnemius cell apoptosis indexes of the metformin group and the HBO group were significantly lower than those of the model group, but higher than those of the control group, with statistical difference ( P<0.05) in all comparison. Conclusion:HBO can improve the skeletal muscle structure of the rats with diabetes, improve the expressions of bFGF and VEGF, decrease the expression of Caspase-3, and reduce cell apoptosis; it, therefore, has a certain protective effect on the skeletal muscle in rats with diabetes.

Objective: To analyze clonal evolution and clinical significance of trisomy 8 in patients with acquired bone marrow failure. Methods: The clinical data of 63 patients with acquired bone marrow failure accompanied with isolated trisomy 8 (+8) from June 2011 to September 2018 were analyzed retrospectively, the clonal evolution patterns and relationship with immmunosuppressive therapy were summarized. Results: Totally 24 male and 39 female patients were enrolled, including 39 patients with aplastic anemia (AA) and 24 patients with relatively low-risk myelodysplastic syndrome (MDS) . Mean size of+8 clone in MDS patients[65% (15%-100%) ]was higher than that of AA patients[25% (4.8%-100%) , z=3.48, P=0.001]. The patients were was divided into three groups (<30%, 30%-<50%,and ≥50%) according to the proportion of+8 clone. There was significant difference among the three groups between AA[<30%:55.6% (20/36) ; 30-50%: 22.2% (8/36) ; ≥50%22.2% (8/36) ]and MDS patients[<30%:19.0% (4/21) ; 30%-<50%:19.0% (4/21) ; ≥50%61.9% (13/21) ] (P=0.007) . The proportion of AA patients with+8 clone <30% was significantly higher than that of MDS patients (P=0.002) ; and the proportion of AA patients with+8 clone ≥50%was significantly lower than that of MDS patients (P=0.002) . The median age of AA and MDS patients was respectively 28 (7-61) years old and 48.5 (16-72) years old. Moreover, there was no correlation between age and+8 clone size in AA or MDS (r_s=0.109, P=0.125; r_s=-0.022, P=0.924, respectively) . There was statistical difference in total iron binding capacity, transferrin and erythropoietin between high and low clone group of AA patients (P=0.016, P=0.046, P=0.012, respectively) , but no significant difference in MDS patients. The immunosuppressive therapy (IST) efficacy of AA and MDS patients was respectively 66.7% and 43.8% (P=0.125) . Comparing with initial clone size (27.3%) , the +8 clone size (45%) of AA patients was increased 1-2 year after IST, but no statistical difference (z=0.83, P=0.272) . Consistently, there was no significant change between initial clone size (72.5%) and 1-2 year clone size (70.5%) after IST in MDS patients. There was no significant difference in IST efficient rate between +8 clone size expansion and decline group of in AA patients at 0.5-<1, 1-2 and>2 years after IST. We found four dynamic evolution patterns of +8 clone, which were clone persistence (45%) , clone disappearance (30%) , clone emergence (10%) and clone recurrence (15%) . Conclusions AA patients had a low clone burden, while MDS patients had a high burden of +8 clone. The +8 clone of AA patients didn’t significantly expanded after IST, and the changes of +8 clone also had no effect on IST response.

Objective: To determine the valuable hemolytic characteristics in differential diagnosis of paroxysmal nocturnal hemoglobinuria (PNH), autoimmune hemolytic anemia (AIHA) and hereditary spherocytosis (HS). Method: The clinical and hemolytic characteristics of 108 PNH patients, 127 AIHA patients and 172 HS patients diagnosed from January 1998 to April 2017 were compared. Results: ①Reticulocyte percentage (Ret%) of PNH patients [6.70% (0.14%-22.82%)] was significantly lower than that of AIHA [14.00%(0.10%-55.95%), P<0.001] and HS patients [11.83%(0.60%-57.39%), P<0.001]. The Ret% in PNH patients were significantly lower than those in AIHA and HS patients at the same levels of anemia, except for in mild anemia between PNH and AIHA patients. However, when comparing the Ret% between AIHA and HS patients, there was significant difference only in mild anemia [7.63%(1.87%-29.20%)% vs 11.20%(3.31%-22.44%), z=-2.165, P=0.030]. ②The level of TBIL in HS patients was significantly higher than that in AIHA and PNH patients [79.3 (11.2-244.0) μmol/L vs 57.6 (7.6-265.0) μmol/L, z=5.469, P<0.001; 79.3(11.2-244.0) μmol/L vs 26.2(4.6-217.7) μmol/L, z=-2.165, P<0.001], and the proportion of HS patients with TBIL more than 4 times the upper limit of normal (ULN) (64.1%) was significantly higher than that of AIHA (37.7%, χ²=19.896, P<0.001) and PNH patients (4.6%, P<0.001). ③The LDH level of PNH patients was significantly higher than that of AIHA and HS [1 500 (216-5 144) U/L vs 487 (29-3 516) U/L, z=-9.556, P<0.001; 1 500 (216-5 144) U/L vs 252 (132-663) U/L, z=-11.518, P<0.001], and the proportion of PNH patients with LDH more than 1 000 U/L (79.1%) was significantly higher than that of AIHA patients (13.0%, χ²=93.748, P<0.001) and HS patients (0, P<0.001). ④Splenomegaly occurred in 43.5% of PNH patients, including 16.0% with severe splenomegaly. In contrast, the occurrence of splenomegaly was 98.6% in AIHA patients and 100.0% in HS patients (P<0.001), and 63.0% of AIHA patients (P<0.001) and 90.4% of HS patients (P<0.001) were with severe splenomegaly. ⑤The prevalence of cholelithiasis in HS patients was up to 43.1%, significantly higher than that in AIHA patients (10.5%, P<0.001) and PNH patients (2.9%, P<0.001). Conclusion The comprehensive assessment of the five hemolytic characteristics is simplified, practical and efficient, with great clinical significance, providing specific indicators for differential diagnosis and efficient approach for making further work-up.

Objective: To analyze the clinical characteristics and long-term outcomes with multicenter study for acute lymphoblastic leukemia (ALL) in children over 10 years old and adolescents. Method: Newly diagnosed ALL patients aged from 10 to 18 years old in three hospitals were included in the study from May 1^st 2005 to April 30^th 2015. They were received ALL-2005/2009 protocol following up to December 31^st 2016. The clinical characteristics, outcomes and the prognostic analysis were evaluated between the two protocols. Results: Totally, 237 patients were involved in the study, 76 cases for ALL-2005 and 161 cases for ALL-2009 protocol. Complete remission (CR) after induction therapy was 94.5%. 64 (28.6%) patients relapsed with a median time of 14.5 months and 70 (29.5%) patients passed away during the following time. In long-term follow-up, the 5-year event-free survival (EFS) and 5-year overall survival (OS) of ALL patients were (63.1±3.3)% and (68.4±3.2)%. The 7-year EFS and OS were (61.0±3.5)% and (67.6±3.3)%.The 5-year EFS of intermediate risk group in ALL-2005 and ALL-2009 protocol were (73.6±6.1)% and (71.7±4.3)% with no difference (χ²=0.064, P=0.801). The 5-year EFS of high risk group in two protocols were (27.6±9.6)% and (33.9±9.3)%, showing no significant difference (χ²=0.296, P=0.586). Five years relapsed rate of two protocols were (33.8±5.7)% and (32.6±4.1)% with no difference (χ²=0.055, P=0.815). The mortalities were 36.8% and 29.8% separately (χ²=2.869, P=0.090). Univariate analysis indicated that age, male, risk, BCR/ABL translocation/t(9;22) and resistant to induction were risk prognostic factors in long-term survival (χ²=4.764, 4.796, 46.410, 9.560, 25.450; P=0.029, 0.029, <0.001, 0.049, <0.001). Cox multivariate analysis showed male, risk and resistant to induction were independent risk prognostic factors (RR=1.790, 2.727, 2.719; P=0.021, 0.000, 0.012). Conclusion Protocol ALL-2009 enhanced the chemotherapy intensity in intermediate risk group with no benefit of survival. BCR-ABL fusion or t(9;22) translocation was still the risk factor of prognosis. TKI inhibitor used in these patients could improve survival. EFS rate was increased a little and death rate was decreased in ALL-2009 protocol with no significant lower relapsed rate comparing with ALL-2005 protocol.