UPLC-Q-Exactive-MS/MS and network pharmacology were employed to preliminarily study the active components and mechanism of Jinwugutong Capsules in the treatment of osteoporosis. Firstly, UPLC-Q-Exactive-MS/MS was employed to characterize the chemical components of Jinwugutong Capsules, and network pharmacology was employed to establish the "drug-component-target-pathway-disease" network. The key targets and main active components were thus obtained. Secondly, AutoDock was used for the molecular docking between the main active components and key targets. Finally, the animal model of osteoporosis was established, and the effect of Jinwugutong Capsules on the expression of key targets including RAC-alpha serine/threonine-protein kinase(AKT1), albumin(ALB), and tumor necrosis factor-alpha(TNF-α) was determined by enzyme-linked immunosorbent assay(ELISA). A total of 59 chemical components were identified from Jinwugutong Capsules, among which coryfolin, 8-prenylnaringenin, demethoxycurcumin, isobavachin, and genistein may be the main active components of Jinwugutong Capsules in treating osteoporosis. The topological analysis of the protein-protein interaction(PPI) network revealed 10 core targets such as AKT1, ALB, catenin beta 1(CTNNB1), TNF, and epidermal growth factor receptor(EGFR). The Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment showed that Jinwugutong Capsules mainly exerted the therapeutic effect by regulating the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT) signaling pathway, neuroactive ligand-receptor interaction, mitogen-activated protein kinase(MAPK) signaling pathway, Rap1 signaling pathway and so on. Molecular docking showed that the main active components of Jinwugutong Capsules well bound to the key targets. ELISA results showed that Jinwugutong Capsules down-regulated the protein levels of AKT1 and TNF-α and up-regulated the protein level of ALB, which preliminarily verified the reliability of network pharmacology. This study indicates that Jinwugutong Capsules may play a role in the treatment of osteoporosis through multiple components, targets, and pathways, which can provide reference for the further research.
Animals ; Tumor Necrosis Factor-alpha/genetics* ; Network Pharmacology ; Capsules ; Molecular Docking Simulation ; Phosphatidylinositol 3-Kinases ; Reproducibility of Results ; Tandem Mass Spectrometry

BACKGROUND: Acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) is an important occurrence in the natural history of idiopathic pulmonary fibrosis (IPF), associated with high hospitalization rates, high mortality and poor prognosis. At present, there is no effective treatment for AE-IPF. Chinese herbal medicine has some advantages in treating IPF, but its utility in AE-IPF is unclear. OBJECTIVE: The treatment of AE-IPF with Kangxian Huanji Granule (KXHJ), a compound Chinese herbal medicine, lacks an evidence-based justification. This study explores the efficacy and safety of KXHJ in patients with AE-IPF. DESIGN, SETTING, PARTICIPANTS AND INTERVENTIONS: We designed a randomized, double-blind, placebo-controlled, exploratory clinical trial. A total of 80 participants diagnosed with AE-IPF were randomly assigned to receive KXHJ or a matching placebo; the treatment included a 10 g dose, administered twice daily for 4 weeks, in addition to conventional treatment. Participants were followed up for 12 weeks after the treatment. MAIN OUTCOME MEASURES: The primary endpoints were treatment failure rate and all-cause mortality. Secondary endpoints included the length of hospitalization, overall survival, acute exacerbation rate, intubation rate, the modified British Medical Research Council (mMRC) score, and the St George's Respiratory Questionnaire for IPF (SGRQ-I) score. RESULTS: The rate of treatment failure at 4 weeks was lower in the intervention group compared to the control group (risk ratio [RR]: 0.22; 95% confidence interval [CI]: 0.051 to 0.965, P = 0.023). There was no significant difference in all-cause mortality at 16 weeks (RR: 0.75; 95% CI: 0.179 to 3.138; P > 0.999) or in the acute exacerbation rate during the 12-week follow-up period (RR: 0.69; 95% CI: 0.334 to 1.434; P = 0.317). The intervention group had a shorter length of hospitalization than the control group (mean difference [MD]: -3.30 days; 95% CI, -6.300 to -0.300; P = 0.032). Significant differences in the mean change from baseline in the mMRC (between-group difference: -0.67; 95% CI: -0.89 to -0.44; P < 0.001) and SGRQ-I score (between-group difference: -10.36; 95% CI: -16.483 to -4.228; P = 0.001) were observed after 4 weeks, and also in the mMRC (between-group difference: -0.67; 95% CI: -0.91 to -0.43; P < 0.001) and SGRQ-I (between-group difference: -10.28; 95% CI, -15.838 to -4.718; P < 0.001) at 16 weeks. The difference in the adverse events was not significant. CONCLUSION: KXHJ appears to be effective and safe for AE-IPF and can be considered a complementary treatment in patients with AE-IPF. As a preliminary exploratory study, our results provide a basis for further clinical research. TRIAL REGISTRATION Chinese Clinical Trial Registry (ChiCTR1900026289). Please cite this article as: Li JS, Zhang HL, Guo W, Wang L, Zhang D, Zhao LM, Zhou M. Efficacy and safety of Kangxian Huanji Granule as adjunctive treatment in acute exacerbation of idiopathic pulmonary fibrosis: an exploratory randomized controlled trial. J Integr Med. 2023; 21(6): 543-549.
Humans ; Double-Blind Method ; Drugs, Chinese Herbal/therapeutic use* ; Idiopathic Pulmonary Fibrosis/drug therapy* ; Treatment Outcome

Objective To explore the protective effects and mechanisms of cysteinyl leukotriene 2(CysLT2)receptor antagonist HAMI3379 on cerebral ischemia injury in rats.Methods 30 male SD rats were randomly divided into sham operation group,model group,and HAMI3379 group,with 10 rats in each group.The rats of model group were subjected to middle cerebral artery occlusion(MCAO)to construct the cerebral ischemia injury model,while HAMI3379 group received intraperitoneal injection of HAMI3379(0.2 mg/kg)before and after MCAO 30 min.The rats after cerebral ischemia injury were scored for neurological symptoms.The infarction volume of rats was observed by TTC staining,the activation marker Iba1 of microglia was detected by immunofluorescence staining,the mRNA level of M1/M2 polarized phenotype molecules of microglia was detected by Real-time PCR,the number of neurons was observed by NeuN staining,and neuronal degeneration was observed by Fluoro-Jade B staining.Western blotting assay was used to detect the expression of CysLT2 protein and nuclear factors κB-related protein Cα(PKCα),IκBα,p65 and p50 proteins in brain tissue.Results Compared with sham operation group,the neurological symptom score and cerebral infarction volume of model group were significantly increased(P<0.05).Compared with model group,the neurological symptom score and cerebral infarction volume of HAMI3379 group were significantly decreased(P<0.05).The results of immunofluorescence staining showed that the expression of microglia activation marker Iba1 was increased in brain tissue of rats after cerebral ischemia injury(P<0.05).Compared with sham operation group,mRNA expression of M1 polarized molecules(CD86,IL-1β,TNF-α)and M2 polarized molecules(CD206,TGF-β,IL-10)were significantly increased in the ischemic central brain tissue of model group(P<0.05).Compared with model group,the expression of M1 polarized molecules in HAMI3379 group was significantly downregulated(P<0.05),while the expression of M2 polarized molecules was significantly upregulated(P<0.05).Compared with sham operation group,the expressions of PKCα,IκBα,p65 and p50 in brain tissue of model group were significantly up-regulated(P<0.05);compared with model group,the expressions of PKCα,IκBα,p65,and p50 in HAMI3379 group were significantly down-regulated(P<0.05).The NeuN staining results showed that the number of neurons in the brain tissue of model group was decreased when compared with sham operation group(P<0.05),while the number of degenerated neurons was increased(P<0.05).Compared with model group,the number of neurons in HAMI3379 group was increased(P<0.05),while the number of degenerated neurons was decreased(P<0.05).Conclusions CysLT2 receptor antagonist HAMI3379 may regulate PKCα/IκBα/NF-κB signaling pathway,inhibits M1 polarization activation of microglia and promotes its transition to M2 polarization,inhibits neuronal degeneration,and plays a neuroprotective role.

Objective:To prepare the hydrogel scaffolds with different concentrations of laponite and compare their osteogenic properties.Methods:The scaffolds of gelatin/sodium alginate hydrogel into which laponite was added according to the mass ratios of 0%, 1%, 2%, and 3% were assigned into groups T0, T1, T2, and T3. In each group, the compressive modulus was measured and the leaching solution for 24 h extracted to measure the ion release. Bone marrow mesenchymal stem cells (BMSCs) were cultured in the extract medium from each group and common medium (blank group) ( n=3) in the in vitro experiments to determine the expression of osteogenic genes Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and type I collagen after 7 days of culture. In the in vivo experiments, the scaffolds were implanted into the femoral condyle defects in rats, and a blank group with no scaffolds was set. The bone repair in each group was evaluated by hematoxylin-eosin(HE) staining and immunohistochemical staining. Results:The compressive modulus in group T2 [(139.05±6.43) kPa] was significantly higher than that in groups T0, T1 and T3 [(68.83±3.76) kPa, (101.18±3.68) kPa and (125.40±3.28) kPa] ( P<0.05). The ion contents of lithium, magnesium and silicon released from the 24 h leaching solution in group T2 were (0.031±0.005) μg/mL, (3.047±0.551) μg/mL and (5.243±0.785) μg/mL, insignificantly different from those in group T3 ( P> 0.05) but significantly larger than those in group T1 ( P>0.05). The in vitro experiments showed that the expression levels of Runx2, ALP and type I collagen in group T2 were 1.59±0.11, 2.02±0.08 and 1.06±0.17, significantly higher than those in the other groups ( P<0.05). HE staining showed that the implanted hydrogel was tightly bound to the bone tissue. Immunohistochemical staining showed that the numbers of Runx2 and osteocalcin positive cells in group T2 were significantly higher than those in the other groups. Conclusions:With ideal biocompatibility, hydrogel scaffolds with different concentrations of laponite can slowly release the decomposed ions of lithium, magnesium and silicon to promote the osteogenic differentiation of BMSCs and the repair of bone defects in vivo. A 2% concentration of laponite in the hydrogel scaffolds may result in the best results.

The advancement of the next generation sequencing (NGS) technology has promoted the development of ancient DNA research. Ancient DNA has made outstanding contributions in various fields such as human origin, animal evolution, etc. How to effectively extract and mine the genetic information from fossil and sub-fossil remains excavated from specific locations is a prerequisite for optimizing their important roles in many fields. In this study, we correlated the two main indicators of DNA damage (terminal base replacement rate, average fragment length) with the possible factors such as the burial time, geological epochs, tissue types, and sequencing library construction methods. The results show that the end base replacement rate of ancient DNA from Northeastern China is positively correlated with the water content of the environment and the ages of the samples. Among samples of different geological epochs, ancient DNA end base replacement rates have significant differences. On the contrary, different tissue types of the remains have no significant effects on the end base replacement rate of ancient DNA. The average fragment size of the molecules has no obvious correlation with the factors mentioned above. The results provide both solid data for investigating the characteristics of ancient DNA from specimens collected in Northeastern China, and valuable information for collecting appropriate samples from different geographical locations and the downstream storage before wet lab procedures after excavation.

Objective:To prepare biomimetic tissue engineering scaffolds of gelatin/sodium alginate/laponite composite hydrogel loaded with BMSCs by 3D biological printing technique，and explore the osteogenic effect of 3D printing on hydrogel scaffolds containing bone marrow mesenchymal stem cells（BMSCs）.Methods:BMSCs were routinely extracted and identified by flow cytometry. Gelatin，sodium alginate and laponite were mixed and then BMSCs were added to prepare cell-containing composite hydrogel scaffolds using 3D bioprinting. Non-printed scaffolds containing cells were prepared by injection molding method. In vitro，the prepared scaffolds were divided into the printing group with cells and non-printing group with cells according to whether they were printed，with 12 samples per group. Another simple cell culture group was set as control. Then，the internal structure of the composite hydrogel was observed by scanning electron microscope，and the expansion rate and water content of the scaffolds were measured by freeze-drying method. At day 3 after culture，the growth status of BMSCs was observed by phalloidine staining. cell counting kit（CCK）-8 assay was used to detect cell activity in scaffolds at days 1，3，and 7 after culture and RT-PCR to detect the expression of osteogenesis related genes Osterix，osteocalcin（OCN）and collagen I at days 7 and 14 ofter culture. In vivo，four groups were set according to printing or not and whether containing cells or not：printing implant group with cells，non-printing implant group with cells，printing implant group without cells and non-printing implant group without cells，with 9 samples per group. Scaffolds in four groups were implanted to the posterior gluteal muscle pouches（random on left or right）of 36 8-week-old SD rats，respectively. The samples were taken X-ray images at 2，4 and 8 weeks after operation，respectively. The osteogenic differentiation of tissues at 8 weeks was observed by HE and Masson staining. Results:The flow cytometry showed that the cells were BMSCs. Internal pores of hydrogels were obvious，and cells stretched freely in the pores. Differences of the swelling rate and water content were not statistically significant between printing group with cells［（1，039.37±30.66）%，（91.21±0.26）%］and non-printing group with cells［（1，032.38±35.05）%，（91.16±0.28）%］（ P>0.05）. At day 3 after culture in vitro，the cells grew well in the hydrogel. After culturing for 1 day in vitro，there was no significant difference in absorbance between printing group with cells and non-printing group with cells（ P>0.05）. At day 3 after culture，there was no significant difference in absorbance between printing group with cells and non-printing group with cells，but both groups showed a higher level than simple cell culture group（ P<0.05）. At day 7 after culture，the absorbance in printing group with cells（2.72±0.17）was higher than that in non-printing group with cells（2.35±0.11），and both of which were higher than that in simple cell culture group（1.95±0.12）（ P<0.05）. At day 7 after culture in vitro，there was no statistically significant difference in the expression of osteogenic differentiation-related genes between printing group with cells and the non-printing group with cells（ P>0.05），but they were all higher than those in simple cell culture group（ P<0.05）. At day 14 after culture in vitro，the expression of osteogenesis-related genes Osterix（1.650±0.095），OCN（2.725±0.091），collagen I（2.024±0.091）in printing group with cells were higher than those in non-printing group with cells（1.369±0.114，2.174±0.198，1.617±0.082，respectively）and those in simple cell culture group（1.031±0.094，1.116±0.092，0.736±0.140，respectively）（ P<0.05）. After implantation for 2 weeks in vivo，with no statistically significant difference in the gray values of X-ray films in each group（ P>0.05）. At weeks 4 and 8 after implantation，the gray values of X-ray films in printing implant group with cells and non-printing implant group with cells were higher than those in printing implant group without cells and non-printing implant group without cells（ P<0.01）. At 8 weeks after implantation，HE staining showed that the scaffolds were degraded in different degrees and immersed with cells，with collagen production seen in Masson staining as well. Conclusions:Composite hydrogel scaffolds can provide a good three-dimensional environment for BMSCs growth. 3D bioprinting can promote the proliferation and osteogenic differentiation of BMSCs in hydrogel scaffolds. In addition，BMSCs-loaded scaffolds can be degraded slowly in vivo with good ectopic osteogenic ability.

OBJECTIVE: To compare the clinical efficacy of distal radius T-plate combined with suture anchor and distal clavicle anatomical locking plate combined with suture anchor in the treatment of Neer Ⅱb distal clavicle fracture. METHODS: From June 2014 to June 2018, 42 patients with Neer Ⅱb distal clavicle fractures were retrospectively analyzed. According to different surgical methods, they were divided into the observation group (T-shaped plate combined with suture anchor) and the control group (anatomical locking plate combined with suture anchor). There were 22 patients in the observation group and 20 patients in the control group. In the observation group, there were 13 males and 9 females, aged from 22 to 70 (45.78± 14.44) years old, 12 cases on the left side and 10 cases on the right side, 8 cases of traffic accident injury and 14 cases of fall. In the control group, there were 12 males and 8 females, aged from 24 to 66 (44.17±15.58) years, 13 cases on the left side and 7 cases on the right side, 6 cases of traffic accident injuryand 14 cases of fall. The operation time, intraoperative blood loss and fracture healing time were compared between the two groups, and Constant Murley score was used to evaluate shoulder joint function. RESULTS: The patients in both groups were followed up for 18 to 24 (20.96±2.02) months. The incisions of both groups were healed at stageⅠ. The fracture ends of both groups were bony healed at the last follow up. There was no significant difference in operation time, intraoperative blood loss and fracture healing time between two groups ( CONCLUSION The two methods can obtain satisfactory results in the treatment of Neer Ⅱb distal clavicle fractures, especially suitable for patients with comminuted distal clavicle fractures or osteoporosis; the clinical effect of the treatment of NeerⅡb distal clavicle fractures with T type distal radius plate combined with suture anchor is satisfactory, which provides another feasible treatment scheme for clinic.
Aged ; Aged, 80 and over ; Bone Plates ; Case-Control Studies ; Clavicle/surgery* ; Female ; Fracture Fixation, Internal ; Fractures, Bone/surgery* ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Suture Anchors ; Treatment Outcome

ObjectiveNano-graphene oxide quantum dots (GOQDs) can be used to target fluorescent markers. The stem cell labeling is an important method in studying stem cell treatments. Our study aims to explore the possibility of using GOQDs as living cell fluorescent marker materials for human periodontal ligament stem cells (hPDLSCs), and to evaluate the biosecurity and effect as live cell fluorescence markers of GOQDs.Methods GOQDs were testified by TEM, DLS, UV-vis, and PL spectra. hPDLSCs were obtained by tissue cultivation and separated by single cell-derived colony selection. Then the source of the cells was carried out by immunocytochemical staining of anti-vimentin, anti-cytokeratin, and multipotent differentiation was used in the identification of stem cells. hPDLSCs were incubated with different concentrations of GOODs (0, 10, 25, and 50 μg/mL) for 24h and 72 h. Cytotoxicity and proliferation effects were determined using CCK-8, and cell cycles were detected using flow cytometry after the co-culture of GOQDs and hPDLSCs. The fluorescent labeling effect of GOQDs was tested using laser scanning confocal microscopy.ResultsThe characterization of GOQDs showed that the nanoparticles were evenly dispersed in water and showing blue light at 365 nm. TEM and DLS showed GOQDs had good dispersion, and the particle size was (6.36±1.41) nm. Immunocytochemical staining of anti-vimentin was positive while anti-cytokeratin was negative. The results of cytotoxicity showed there were no significant differences in cell activity after incubated with different concentrations of GOODs (0, 5, 10, 25, 50, 100, 200, and 400 μg/mL) (P>0.05), and there was no significant decrease in cell activity between 24h and 72h (P>0.05). There was no significant difference in the proportional distribution of G1, G2, and S phases between the two concentrations of GOQDs (0 μg/mL and 50 μg/mL) (P>0.05). Fluorescent images showed that GOQDs could enter the cell membrane and increase the fluorescence intensity at the concertation of 50 μg/mL.ConclusionGOQDs were confirmed to have good biocompatibility and could be used for live cell labeling of hPDLSCs.

Purpose To investigate the expression and clinical significance of free fatty acid receptor 4 (FFAR4) in hepatocellular carcinoma (HCC) . Methods The expression of FFAR4 in HCC tissues and adjacent tissues of HCC patients was confirmed by 102 cases of liver resection and postoperative pathology, and the relationship between FFAR4 expression and clinical data of HCC patients was analyzed. Quantitative realtime PCR (qRT-PCR) and Western blot were used to detect the expression of FFAR4 in 20 pairs of freshly frozen HCC and adjacent tissues,and the related literatures were reviewed. Results The expression rate of FFAR4 in HCC tissues was 64. 7％ (66/102) ,and that in adjacent tissues was 15. 7％ (16/102) . The difference in FFAR4 expression between the two groups was statistically significant (P < 0. 05) . The high expression of FFAR4 in HCC tissues was significantly correlated with tumor vascular invasion (P < 0. 05) ,TNM stage (P < 0. 01) ,and Edmondson classification (P < 0. 05) . qRT-PCR and Western blot showed that the expression of FFAR4 in HCC tissues was significantly higher than that in adjacent tissues. The difference between the two groups was statistically significant (P < 0. 01,P< 0. 05) . Conclusion The expression of FFAR4 is significantly associated with the presence of vascular invasion,TNM staging, and Edmondson grading in HCC. High expression of FFAR4 may be closely related to the severity of HCC patients.

Objective To investigate drug resistance genes and epidemic characteristics of β-lactamase carried by carbapenem-resistant Acinetobacter baumannii (CRAB) in the respiratory intensive care unit(RICU) in a hospital.Methods Clinically isolated CRAB from RICU patients in October-December 2015 were collected.Five drug resistance genes (KPC-2,IMP,VIM,NDM-1,OXA-23) were specifically amplified by polymerase chain reaction (PCR),amplified products were performed agarose gel electrophoresis and sequencing analysis,the homology was analyzed with pulsed-field gel electrophoresis (PFGE).Results A total of 22 CRAB strains were isolated in October-December 2015,19 (86.36％) of which were isolated from sputum.The resistance rate of 22 CRAB strains to compound sulfamethoxazole was 59.09 ％,resistance rate to minocycline was 9.09 ％,all were sensitive to polymyxin B,resistance rates to other antimicrobial agents were more than 80％.Three kinds of resistance genes KPC-2,IMP and NDM-1 were not found by PCR amplification,positive rates of VIM and OXA-23 were both 100％.PFGE homology analysis revealed that 22 strains were divided into 13 different types,each type contained 1-5 strains,9 types(69.23％) contained only 1 strain respectively,the other 4 types (30.77％) contained 2-5 strains.A5,A7,and A8;A9,A11,A14,A19 and A22;A4,A10 and A12;A16 and A18 were of the same type respectively.Conclusion The main types of β-lactamase-resistant genes of CRAB in RICU are VIM and OXA-23.Homology analysis shows a small parts are of the same clone strains,which reveals epidemic of a small scale.