The value of toxicological analysis in acute poisoning patients with uncertain exposure histories: a retrospective and descriptive study from an institute of poisoning.

BACKGROUND: In clinical practice, some patients might not be able or unwilling to provide a thorough history of medication and poison exposure. The aim of this study was to use toxicological analysis to examine the clinical characteristics of patients with acute poisoning whose exposure history was uncertain from a toxicological analysis perspective. METHODS: This was a retrospective and descriptive study from an institute of poisoning. Patient registration information and test reports spanning the period from April 1, 2020 to March 31, 2022, were obtained. Patients with uncertain exposure histories and who underwent toxicological analysis were included. Clinical manifestations and categories of toxics were analyzed. RESULTS: Among the 195 patients with positive toxicological analysis results, the main causes of uncertain exposure history was disturbance of consciousness (62.6%), unawareness (23.6%) and unwillingness or lack of cooperation (13.8%). The predominant clinical manifestations were disturbed consciousness (62.6%), followed by vomiting and nausea (14.4%) and liver function abnormalities (8.7%). A comparison of clinical manifestations between patients with positive and negative (n=99) toxicological analyses results revealed significantly different proportions of disturbances in consciousness (63% vs. 21%), dizziness (1.5% vs. 5.1%), multi-organ failure (1.5% vs. 7.1%), and local pain (0 vs 4%). The main categories of substances involved were psychiatric medications (23.1%), sedatives (20.5%), insecticides (13.8%), and herbicides (12.8%). CONCLUSION: The clinical manifestations of acute poisoning in patients with an uncertain exposure history are diverse and nonspecific, and toxicological analysis plays a pivotal role in the diagnosis and differential diagnosis of such patients.

Non-cytochrome P450 enzyme aldehyde oxidase is involved in the oxidative metabolic pathway of diquat and its detoxification effect.

Diquat (DQ) poisoning has garnered attention in recent years, primarily due to the rising incidence of cases worldwide, coupled with the absence of a viable antidote for its treatment. Despite the fact that diquat monopyridone (DQ-M) has been identified as a significant metabolite of DQ, the enzyme responsible for its formation remains unknown. In this study, we have identified aldehyde oxidase (AOX) as a vital enzyme involved in DQ oxidative metabolism. The metabolism of DQ to DQ-M was significantly inhibited by AOX inhibitors including raloxifene and hydralazine. The source of oxygen incorporated into DQ-M was proved to be from water through a H218O incubation experiment which further corroborated DQ-M formation via AOX metabolism. The product of DQ-M in vitro generated by fresh rat tissues co-incubation was consistent with its AOX expression. The result of the molecular docking analysis of DQ and AOX protein showed that DQ is capable of binding to AOX. Furthermore, the cytotoxicity of DQ was significantly higher than DQ-M at the same concentration tested in six cell types. This work is the first to uncover the involvement of aldehyde oxidase, a non-cytochrome P450 enzyme, in the oxidative metabolic pathway of diquat, thus providing a potential target for the development of detoxification treatment.

Unilateral Bi/Multi-Portal Endoscopy for the Treatment of Complicated Lumbar Degenerative Diseases with Utilization of Uniaxial Spinal Endoscope, Instead of Arthroscope: Technique Note and Clinical Results.

Objective: This article aims to discuss a novel surgical strategy, referred to as unilateral bi/multi-portal endoscopy (UME), which used a uniaxial spinal endoscope instead of an arthroscope in the traditional unilateral biportal endoscopy (UBE) surgical procedure in our study of the treatment of complicated lumbar degenerative diseases. Methods: This retrospective study included 42 patients diagnosed with high-migrated lumbar disc herniation and bilateral spinal stenosis who underwent UME surgery from January 2021 to December 2021. Patients included 20 men and 22 women, with an average age of 55.97±14.92 years. The average follow-up period was 13.19 months. The demographic data, operation time (min), and complications were recorded and analyzed. The visual analogue scale (VAS), Oswestry Disability Index (ODI) scores were used to evaluate the surgical outcomes. Three-dimensional CT scans and MRI were conducted to evaluate the radiographic improvement. Results: A total of 26 patients were diagnosed with lumbar disc herniation and 16 with lumbar spinal stenosis. All 42 patients underwent UME surgery and achieved satisfactory outcomes. The operation time was 154.46±46.09 min. The average follow-up time was 13.19±1.33 months. The preoperative back pain (VAS-Back) and the last follow-up VAS-Back were 3.84±1.00 and 0.70±0.46, respectively (P < 0.05). The preoperative leg pain (VAS-Leg) and the last follow-up VAS-Leg were 6.46±1.08 and 1.03±0.64, respectively (P <0.05). Significant differences existed between preoperative ODI scores (58.70±11.22%) and the last follow-up ODI scores (9.24±3.04%; P<0.05). All patients achieved significant pain relief and functional improvement after the surgery. No severe complications occurred, except for two cases of postoperative dysesthesia and one case suffered from vertebral compression fractures induced by a postoperative accidental injury. Symptoms of numbness disappeared within one week with treatment using dexamethasone and neurotrophic drugs. The vertebral fracture case recovered with percutaneous kyphoplasty treatment. Conclusion: This study suggests that UME is a promising treatment strategy for high-migrated disc herniation and bilateral spinal stenosis.

Lomatogonium rotatum extract alleviates diabetes mellitus induced by a high-fat, high-sugar diet and streptozotocin in rats.

BACKGROUND: Lomatogonium rotatum (LR) is traditionally used in Mongolian folk medicine as a hypoglycemic agent, but its evidence-based pharmacological effects and me-chanisms of action have not been fully elucidated. AIM: To emphasize the hypoglycemic action mechanism of LR in a type 2 diabetic rat model and examine potential biomarkers to obtain mechanistic understanding regarding serum metabolite modifications. METHODS: A high-fat, high-sugar diet and streptozotocin injection-induced type 2 diabetic rat model was established. The chemical composition of the LR was identified by high performance liquid chromatography. LR extract administrated as oral gavage at 0.5 g/kg, 2.5 g/kg, and 5 g/kg for 4 wk. Anti-diabetic effects of LR extract were evaluated based on histopathological examination as well as the measurement of blood glucose, insulin, glucagon-like peptide 1 (GLP-1), and lipid levels. Serum metabolites were analyzed using an untargeted metabolomics approach. RESULTS: According to a chemical analysis, swertiamarin, sweroside, hesperetin, coumarin, 1.7-dihydroxy-3,8-dimethoxyl xanthone, and 1-hydroxy-2,3,5 trimethoxanone are the principal active ingredients in LR. An anti-diabetic experiment revealed that the LR treatment significantly increased plasma insulin and GLP-1 levels while effectively lowering blood glucose, total cholesterol, triglycerides, low-density lipoprotein cholesterol, and oral glucose tolerance test compared to the model group. Furthermore, untargeted metabolomic analysis of serum samples detected 236 metabolites, among which 86 were differentially expressed between the model and the LR group. It was also found that LR considerably altered the levels of metabolites such as vitamin B6, mevalonate-5P, D-proline, L-lysine, and taurine, which are involved in the regulation of the vitamin B6 metabolic pathway, selenium amino acid metabolic pathway, pyrimidine metabolic pathway, and arginine and proline metabolic pathways. CONCLUSION: These findings indicated that LR may have a hypoglycemic impact and that its role may be related to changes in the serum metabolites and to facilitate the release of insulin and GLP-1, which lower blood glucose and lipid profiles.

[Anti-obesity and lipid-lowering mechanism of Corydalis Bungeanae Herba: based on intestinal microflora and metabolomics].

This study aims to explore anti-obesity and lipid-lowering mechanism of Corydalis Bungeanae Herba(CB) based on intestinal microflora and metabolomics. Specifically, high-fat high-sugar diet(HFHS, 10 weeks) was used to induce obesity in rats. Then the model rats were randomized into the model group, low-dose(0.18 g·kg~(-1)), medium-dose(0.9 g·kg~(-1)), and high-dose(1.8 g·kg~(-1)) CBH groups, and orlistat group(0.03 g·kg~(-1)), 12 in each group. Rats which received normal diet were used as control. The body weight and feed intake of rats were recorded every week. After 6 weeks of administration, rats were killed and gastric emptying and small intestinal propulsion were examined. Enzyme-linked immunosorbent assay(ELISA) was employed to analyze serum indexes, and liver and perirenal fat were collected for haematoxilin-eosin(HE) staining. Rat feces and serum were gathered for 16 S rDNA sequencing and metabolomics analysis and Spearman's correlation analysis was performed to explore the correlation between differential microflora and differential metabolites. The result showed that CBH extract decreased body weight, feed intake, and serum cholecystokinin(CCK), triglyceride(TG), and total cholesterol(TC), delayed gastric emptying, and reduced fat accumulation in liver and perirenal adiposity as compared with rats in the model group. In addition, Lachnospiraceae and Sutterellaceaecan significantly decreased in the model group, but CBH extract up-regulated their abundance. Moreover, the abundance of Prevotellaceae was significantly raised by HFHS, but CBH decreased it. Glutaric acid, glyceric acid, hippuric acid, malic acid, glyceric acid, oxoglutaric acid, fumaric acid/succinic acid, oxoglutaric acid/isocitric acid, D-glucuronic acid, cholic acid were the main deferentially expressed metabolites and significantly correlated with Sutterellaceae and Prevotellaceae. These key metabolites and microbiota mainly involved in tricarboxylic acid(TCA) cycle, glucose metabolism, amino acid metabolism, and energy metabolism. This study proved that CBH can efficiently improve body weight and blood lipids, reduce adipocyte volume, and positively regulate the intestinal microflora and serum metabolites, thereby achieving the anti-obesity and lipid-owering effect.

Antitumor effects of the small molecule DMAMCL in neuroblastoma via suppressing aerobic glycolysis and targeting PFKL.

BACKGROUND: Neuroblastoma (NB) is a common solid malignancy in children that is associated with a poor prognosis. Although the novel small molecular compound Dimethylaminomicheliolide (DMAMCL) has been shown to induce cell death in some tumors, little is known about its role in NB. METHODS: We examined the effect of DMAMCL on four NB cell lines (NPG, AS, KCNR, BE2). Cellular confluence, survival, apoptosis, and glycolysis were detected using Incucyte ZOOM, CCK-8 assays, Annexin V-PE/7-AAD flow cytometry, and Seahorse XFe96, respectively. Synergistic effects between agents were evaluated using CompuSyn and the effect of DMAMCL in vivo was evaluated using a xenograft mouse model. Phosphofructokinase-1, liver type (PFKL) expression was up- and down-regulated using overexpression plasmids or siRNA. RESULTS: When administered as a single agent, DMAMCL decreased cell proliferation in a time- and dose-dependent manner, increased the percentage of cells in SubG1 phase, and induced apoptosis in vitro, as well as inhibiting tumor growth and prolonging survival in tumor-bearing mice (NGP, BE2) in vivo. In addition, DMAMCL exerted synergistic effects when combined with etoposide or cisplatin in vitro and displayed increased antitumor effects when combined with etoposide in vivo compared to either agent alone. Mechanistically, DMAMCL suppressed aerobic glycolysis by decreasing glucose consumption, lactate excretion, and ATP production, as well as reducing the expression of PFKL, a key glycolysis enzyme, in vitro and in vivo. Furthermore, PFKL overexpression attenuated DMAMCL-induced cell death, whereas PFKL silencing promoted NB cell death. CONCLUSIONS: The results of this study suggest that DMAMCL exerts antitumor effects on NB both in vitro and in vivo by suppressing aerobic glycolysis and that PFKL could be a potential target of DMAMCL in NB.

Handgrip strength is positively associated with bone mineral density in middle and aged adults: results from NHANES 2013-2014.

Few studies have examined the association between handgrip strength and BMD in specific subgroups. Therefore, we examined the associations of handgrip strength with BMD aged ≥ 40 years and found that handgrip strength is associated with BMD which is independent of BMI, physical activity, and other potential confounders. PURPOSE: Previous studies have revealed that handgrip strength is a measure of muscular fitness and is associated with fracture and bone mineral density (BMD) in adolescents and adults, with conflicting results. In addition, few studies have examined the association between handgrip strength in predefined subgroups such as sex, age, and physical activity in a whole population. METHODS: We examined the associations of handgrip strength with BMD in 2720 adults (1359 men and 1361 women) aged ≥ 40 years (mean age, 58.6 ± 11.8 years) from the National Health and Nutrition Examination Survey (NHANES) 2013-2014. NHANES collects data via household interviews and direct standardized physical examinations conducted in specially equipped mobile examination centers. The date of final data collection was 2014 and the present data analysis was conducted in January to February 2020. RESULTS: Handgrip strength was significantly associated with total femur (r = 0.482, P < 0.001) and femoral neck BMD (r = 0.427, P < 0.001) among all participants, respectively. After adjustment for age, sex, race, body mass index (BMI), physical activity, smoking, history of diabetes, history of hypertension, and history of high cholesterol, each unit (1 SD) of BMI-adjusted handgrip strength was positively associated with 0.026 g/cm2 increase in total femur BMD (P < 0.001) and 0.027 g/cm2 increase in femoral neck BMD (P < 0.001). There was a significant increasing trend in total femur and femoral BMD as handgrip strength increased from the lowest quartile to the highest quartile (P for trend < 0.001). For subgroup analysis, there were no significant interaction effects of handgrip strength with BMD between predefined subgroups (all P > 0.05). CONCLUSIONS: High level of handgrip strength is associated with increased BMD. The association is independent of BMI, physical activity, and other potential confounders.

Protective effect and mechanisms of action of Mongolian medicine Sulongga-4 on pyloric ligation-induced gastroduodenal ulcer in rats.

BACKGROUND: Sulongga-4 (SL-4) is a herbal formula used in traditional Mongolian medical clinics for the treatment of peptic ulcers and gastroenteritis, even though its pharmacological mechanism has not been well characterized. AIM: To evaluate the protective effect and identify the mechanisms of action of SL-4 on gastroduodenal ulcer induced by pyloric ligation (PL) in rats. METHODS: PL was performed to induce gastric and duodenal ulcers in rats, which were then treated with oral SL-4 (1.3, 2.6, or 3.9 g/kg per day) for 15 d. PL-induced gastroduodenal ulceration. Therapeutic effects were characterized by pathological and histological evaluations and inflammatory indicators were analyzed by enzyme-linked immunosorbent assay. Microarray analyses were conducted to identify gene expression profiles of gastroduodenal tissue in PL rats with or without SL-4 treatment. The candidate target genes were selected and verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR). RESULTS: SL-4 decreased histopathological features in the PL-induced ulcerated rats. SL-4 significantly (P < 0.05) decreased expression of tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, endotoxin, platelet-activating factor, and increased prostaglandin E2 and epidermal growth factor in ulcer tissue. Microarray analysis was used to identify a panel of candidate target genes for SL-4 acting on PL-induced ulceration. Genes included some complement and coagulation cascade and retinol metabolism pathways that are closely associated with inflammatory responses and gastric mucosal protective mechanisms. qRT-PCR showed that altered expression of the selected genes, such as CYP2b2, UGT2b1, A2m, and MASP1 was consistent with the microarray results. CONCLUSION: SL-4 exerts protective effects against PL-induced gastroduodenal ulcers via reducing inflammatory cytokines and elevating expression of gastric acid inhibitory factors. Downregulation of CYP2b2 and UGT2b1 genes in retinol metabolism and upregulation of A2m and MASP1 genes in the complement and coagulation cascades pathways are possibly involved in SL-4-mediated protection against gastroduodenal ulcer.

Novel agent DMAMCL suppresses osteosarcoma growth and decreases the stemness of osteosarcoma stem cell.

Osteosarcoma (OS) is the most common primary malignancy of bone that mostly affects children, adolescents, and young people. Despite advances have been made in multimodal therapy of OS, the long-term survival rate has reached a plateau, and the main obstacles are bad response to chemotherapy and gained chemoresistance. In this study, we tested the therapeutic effect of a newly reported drug, DMAMCL, on OS. Five human OS cell lines (143B, MNNG, MG63, Saos-2, U-2OS), and the mouse fibroblast cell line (NIH3T3) and human retinal epithelial cell (ARPE19) were used. The anti-tumor effect of DMAMCL was studied by MTS assay or IncuCyte-Zoom (in vitro), and Xenograft-mice-model (in vivo). Changes of cell cycle, apoptotic cells, caspase3/7 activities, and stemness after DMAMCL treatment were investigated. BAX siRNAs were used to knockdown the expression of BAX. Expressions of CyclinB1, CDC2, BCL-2 family, PARP, CD133, and Nanog were measured by Western Blotting. DMAMCL-induced dose-dependent OS cell death in vitro, and suppressed tumor growth and extended the survival of xenograft-bearing mice. DMAMCL-induced G2/M phase arrest in vitro, and apoptosis both in vitro and in vivo. Down-regulation of BAX expression attenuated the DMAMCL-induced OS cell death in vitro. We also found that DMAMCL inhibited the stemness in OS cells. These results indicated that DMAMCL possess therapeutic value in OS and may be a promising candidate for the new drug discovery for OS therapy.

Activating transcription factor 6 (ATF6) negatively regulates Polo-like kinase 4 expression via recruiting C/EBPß to the upstream-promoter during ER stress.

Polo-like kinase 4 (PLK4) is a member of the serine/threonine protein kinase family involved in cell-cycle regulation and cellular response to stresses. However, the alteration of PLK4 in response to endoplasmic reticulum (ER) stress has not been well described. In the present study, we focused on the regulation of PLK4 regulation in response to ER stress. PLK4 expression was dramatically reduced under ER stress induced by brefeldin A (BFA), tunicamycin (TM), or thapsigargin (TG) and down regulation of PLK4 expression was dependent on activating transcription factor 6 (ATF6) and CCAAT/enhancer-binding protein ß (C/EBPß). Luciferase activity analysis of the truncated PLK4 promoter indicated that region from -1343 to -1250 of the PLK4 promoter was sensitive to BFA or TG. Additionally, ChIP and ChIP Re-IP assays showed that ATF6 and C/EBPß were assembled on the same region of Plk4 promoter. Notably, we identified one C/EBPß responsive element at position -1284, to which ATF6 or C/EBPß binding was enhanced by BFA or TG under in vitro and in vivo conditions. Finally, overexpression of PLK4 inhibits apoptosis and promotes cell proliferation in response to ER stress. In summary, these results demonstrated that ER stress plays a crucial role in PLK4 expression. ATF6 may upregulate DNA-binding affinities after BFA treatment, via recruiting C/EBPß to the upstream promoter of PLK4. These findings may contribute to the understanding of the molecular mechanism of PLK4 regulation.

Cyclic GMP-AMP synthase is essential for cytosolic double-stranded DNA and fowl adenovirus serotype 4 triggered innate immune responses in chickens.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a predominant DNA sensor inducing the activation of the innate immune responses that produce proinflammatory cytokines and type I interferons, which has been well-investigated in mammals. However, chicken cGAS (chcGAS), which participates in avian innate immunity, has not been well-investigated. Here, we cloned the complete open reading frame sequence of chcGAS. Multiple sequence alignment and phylogenetic analysis revealed that chcGAS was homologous to mammalian cGAS. The chcGAS mRNA was highly expressed in the bone marrow and ileum. The subcellular localization of chcGAS was mainly in the cytoplasm, and partial co-localization was observed in the endoplasmic reticulum. Through overexpression and RNA interference, we demonstrated that chcGAS responded to exogenous dsDNA, HS-DNA, and poly(dA:dT), and to self dsDNA from the DNA damage response, thereby triggering the activation of STING/TBK1/IRF7-mediated innate immunity in both chicken embryonic fibroblasts and chicken liver cancer cells. Furthermore, downregulation of chcGAS enhanced the infection of fowl adenovirus serotype 4 in LMH cells. Our results demonstrated that chcGAS was an important cytosolic DNA sensor activating innate immune responses and may shed light on a strategy for preventing infectious diseases in the poultry industry.

Relationships Between Circulating Irisin Response to Ice Swimming and Body Composition in People With Regular Exercise Experience.

Severe cold exercise involves the irisin response, and may be related to body composition. We aimed to investigate changes in circulating irisin after ice swimming (IS), as well as to evaluate the correlation between body composition and the change in irisin caused by IS (Δirisin). 81 ice swimmers were recruited to perform IS activities. Blood samples were drawn 30 min before and 30 min after IS, and the serum levels of irisin and the ice swimmers' body composition were measured. As results, circulating irisin declined significantly during the recovery period following IS exercise (P < 0.001). The afternoon baseline circulating irisin level and Δirisin in response to IS were correlated with body fat characteristics rather than muscle parameters in ice swimmers. Δirisin subgroup analyses showed that the Δirisin ascending group (Δirisin+) subjects had a higher fat composition and higher basal irisin levels than the Δirisin descending group (Δirisin-). Furthermore, the decrease in irisin was negatively correlated with fat components in Δirisin- subjects, whereas no correlation was observed between the increase in irisin and body composition in Δirisin + subjects. Finally, a non-linear association analysis suggested that body fat indicators had obvious curvilinear relationships with Δirisin. In conclusion, IS caused a significant decrease in irisin. Statistical and curvilinear associations suggested that the correlation between fat tissue and Δirisin caused by IS is dimorphic and the underlying mechanisms may be due to the different metabolic states of subjects.

The anti-tumor growth effect of a novel agent DMAMCL in rhabdomyosarcoma in vitro and in vivo.

BACKGROUND: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children with poor survival. New treatment approaches are urgently needed to improve treatment efficacy in RMS patients. DMAMCL is a novel agent from Asteraceae family that has been tested in phase I clinical trials in adult glioma in Australia. METHODS: Five RMS cell lines (RD, RH18, RH28, RH30 and RH41) were used. The in vitro anti-tumor effect of DMAMCL, alone or in combination with VCR or Epirubicin, was studied using MTS assay or IncuCyte-Zoom cell confluency assay, and further validated by xenograft-mouse model in vivo. Changes in caspase-3/7 activity, cell-cycle progression and generation of ROS after DMAMCL treatment were investigated. Bim mRNA expression was measured by RT-qPCR, and protein expressions of Bim and phosphorylated-NF-κB(p65) by Western blotting. Small interfering RNAs (siRNA) of Bim were used to study the role of Bim in DMAMCL-induced cell death. RESULTS: In vitro, DMAMCL treatment induced a dose-dependent increase in cell death that could be blocked by pan-caspase-inhibitor-Z-VAD-fmk in five RMS cell lines. The percent of cells in SubG1 phase and activities of caspase-3/7 increased after DMAMCL treatment; The combination of DMAMCL with VCR or Epirubicin significantly increased cell death compared to each reagent alone. In vivo, DMAMCL(75 mg/kg or 100 mg/kg) inhibited tumor growth and prolonged survival of mice bearing xenograft RMS tumors (RD, RH18, RH30, RH41). Compared to treatment with DMAMCL or VCR, a combination of two reagents caused significant inhibition of tumor growth (RD, RH41), even after treatment termination. The expression of Bim increased at protein level after DMAMCL treatment both in vitro and in vivo. The expression of p-NF-κB(p65) had a transient increase and the generation of ROS increased after DMAMCL treatment in vitro. Transfection of Bim siRNA into RMS cells blocked the DMAMCL-induced increase of Bim and partially attenuated the DMAMCL-induced cell death. CONCLUSION: DMAMCL had an anti-tumor growth effect in vitro and in vivo that potentially mediated by Bim, NF-κB pathway and ROS. A combination of DMAMCL with chemotherapeutic drugs significantly increased the treatment efficacy. Our study supports further clinical evaluation of DMAMCL in combination with conventional chemotherapy.

[Fingerprinting and multi-indicator quantitative analysis of Mongolian drug Digeda-4 decoction].

To establish the high performance liquid chromatography (HPLC) fingerprint for Digeda-4 decoction (DGD-4D), determine the contents of aesculetin, geniposide, picroside Ⅰ, picroside Ⅱ and ellagicacid in DGD-4D, and provide the scientific foundation for quality control of DGD-4D. The analysis was performed on Diamonsil(2) C18 (4.6 mm×250 mm,5 µm) column, with methanol-0.1% phosphoric acid aqueous solution as mobile phase for gradient elution. The flow rate was 1.0 mL·min⁻¹; injection size was 10 µL; temperature was maintained at 30 °C, and the detection wavelength was set at 254 nm. The common mode of DGD-4D HPLC fingerprint was established, and the hidden information was analyzed by Chemometrics. Chromatographic peaks for DGD-4D were identified by HPLC and quantitative analysis was conducted for characteristic peaks. There were 17 common peaks in the fingerprints and the similarity of the fingerprints was over 0.9 in all 15 batches. The samples were broadly divided into four kinds by principal component analysis and clustering analysis. Four marker compounds were verified by partial least squares discriminant analysis, and No. 9, 12 and 14 peaks were identified as geniposide, picroside Ⅱ, and picroside Ⅰ respectively. The average recoveries were in the range of 95.91%-97.31%. The HPLC fingerprint method for content determination is reliable, accurate, rapid, simple, and reproducible, and can be used as one of the effective methods to control the quality of DGD-4D.

Myostatin knockout induces apoptosis in human cervical cancer cells via elevated reactive oxygen species generation.

Myostatin (Mstn) is postulated to be a key determinant of muscle loss and cachexia in cancer. However, no experimental evidence supports a role for Mstn in cancer, particularly in regulating the survival and growth of cancer cells. In this study, we showed that the expression of Mstn was significantly increased in different tumor tissues and human cancer cells. Mstn knockdown inhibited the proliferation of cancer cells. A knockout (KO) of Mstn created by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 9 (CRISPR/Cas9) induced mitochondria-dependent apoptosis in HeLa cells. Furthermore, KO of Mstn reduced the lipid content. Molecular analyses demonstrated that the expression levels of fatty acid oxidation-related genes were upregulated and then increased rate of fatty acid oxidation. Mstn deficiency-induced apoptosis took place along with generation of reactive oxygen species (ROS) and elevated fatty acid oxidation, which may play a role in triggering mitochondrial membrane depolarization, the release of cytochrome c (Cyt-c), and caspase activation. Importantly, apoptosis induced by Mstn KO was partially rescued by antioxidants and etomoxir, thereby suggesting that the increased level of ROS was functionally involved in mediating apoptosis. Overall, our findings demonstrate a novel function of Mstn in regulating mitochondrial metabolism and apoptosis within cancer cells. Hence, inhibiting the production and function of Mstn may be an effective therapeutic intervention during cancer progression and muscle loss in cachexia.

Leonurine hydrochloride promotes osteogenic differentiation and increases osteoblastic bone formation in ovariectomized mice by Wnt/ß-catenin pathway.

Leonurine hydrochloride (LH) is a synthetic chemical compound derived from leonurine that can be extracted from Leonurus sibiricus and possesses antioxidant, anti-apoptosis, and neuroprotective activities. In previous studies, LH has been demonstrated to attenuate osteoclast activity and prevent bone loss. However, it is unknown whether LH accelerates bone formation and promotes osteogenic differentiation. We systematically examined the effects of LH on ovariectomized-induced osteoporotic mice and the MC3T3-E1 osteoblastic cell line. The results revealed that LH enhanced differentiation of MC3T3-E1 cells, with a dose-dependent increase in alkaline phosphatase (ALP) activity. Moreover, LH upregulated osteogenesis-related gene expression, including osterix, alpha 1 type 1 collagen, runt-related transcription factor 2 (Runx2) and ALP, as shown by quantitative reverse transcription-polymerase chain reaction analysis. At the same time, elevated expression of low-density lipoprotein receptor-related protein 5 and ß-catenin mRNA was detected in the Wnt/ß-catenin pathway. A western blot analysis revealed that LH dose-dependently increased the expression of Runx2 and ß-catenin, and promoted phosphorylation of glycogen synthase kinase-3ß in vitro. The in vivo results showed that administering LH (15 mg/kg/d) for 8 weeks alleviated destruction of the trabecular microstructure caused by osteoporosis. LH increased the bone mineral density and trabecular number, decreased trabecular separation according to a micro-computed tomography scan. In addition, LH enhanced the expression of ß-catenin and Runx2 in vivo. In conclusion, LH promoted osteogenic differentiation and bone formation in vivo and in vitro, which alleviated osteoporosis through activation of the Wnt/ß-catenin pathway.