Differential metabolite analysis of the pharmacodynamic differences between different ratios of Dahuang ()-Taoren () herb pair.

OBJECTIVE: To explore the material basis of the difference of efficacy of Dahuang (Radix Et Rhizoma Rhei Palmati)-Taoren (Semen Persicae) (DT) drugs with different proportions. METHODS: Samples of different ratios of Dahuang (Radix et Rhizoma Rhei Palnati, DH) to Taoren (Semen Persicae, TR) (Group A 1:1, B 2:3, C 3:2) were analyzed based on gas chromatography time-of-flight mass spectrometry untargeted metabolomics technique. RESULTS: A total of 240 primary metabolites were detected. Forty-one differential metabolites involved nine differential metabolic pathways, of which four were closely related to the efficacy of DT in the treatment of heat and blood stasis syndrome. These pathways included the biosynthesis of amino acid (phenylalanine tyrosine and tryptophan), flavonoids, unsaturated fatty acids, and the glycolysis/glycogenesis pathway. CONCLUSION: There are significant differences in the efficacy of different ratios of DT drugs, and their optimal ratio for the treatment of heat and blood stasis syndrome should be 1:1.

Two-dimensional liquid chromatography measurement of meropenem concentration in synovial fluid of patients with periprosthetic joint infection.

Periprosthetic joint infection (PJI) is a catastrophic complication following joint replacement surgery. One potential treatment approach for PJI could be the combination of one-stage revision and intra-articular infusion of antibiotics. Meropenem is one of the commonly used intra-articular antibiotics in our institution. Determining the concentration of meropenem in the joint cavity could be crucial for optimizing its local application, effectively eradicating biofilm infection, and improving PJI treatment outcomes. In this study, we developed a simple, precise, and accurate method of two-dimensional liquid chromatography (2D-LC) for determining the concentration of meropenem in human synovial fluid. The method was then validated based on the guidelines of the Food and Drug Administration and the Chinese Pharmacopoeia. Meropenem showed good linearity in the range of 0.31-25.01 µg/mL (r ≥ .999). Selectivity, intra-day and inter-day precision and accuracy, extraction recovery, and stability validation results were all within the acceptance range. This method has been successfully applied to the determination of synovial fluid samples from PJI patients, providing a useful detection method for meropenem therapeutic drug monitoring (TDM) in PJI patients.

Prolonged duration of epidural labour analgesia decreases the success rate of epidural anaesthesia for caesarean section.

OBJECTIVE: To summarise the process of conversion of epidural labour analgesia to anaesthesia for caesarean delivery and explore the relationship between duration of labour analgesia and conversion. METHODS: Parturients who underwent conversion from epidural labour analgesia to anaesthesia for caesarean delivery between May 2019 and April 2020 at the Chengdu Women's and Children's Central Hospital, Sichuan Maternal and Child Health Hospital, and Jinjiang District Maternal and Child Health Hospital were selected. If the position of the epidural catheter was correct and the effect was good, patients were converted to epidural surgical anaesthesia. If epidural labour analgesia was ineffective, spinal anaesthesia (SA) was administered immediately. For category-1 emergency caesarean sections, general anaesthesia (GA) was administered. RESULTS: A total of 1084 parturients underwent conversion. Of these, 19 (1.9%) received GA due to the initiation of category-1 emergency caesarean section. 704 (64.9%) were converted to epidural surgical anaesthesia, 2 (0.2%) had failed conversions and were administered GA before delivery, and 357 (32.9%) were converted to SA. Logistic regression analysis showed that prolonged duration of epidural labour analgesia ([Crude odds ratio (OR)=1.065; 95% confidence interval (CI), 1.037-1.094; p < .01]; [Adjusted OR = 1.060; 95% CI, 1.031-1.091; p < .01]) was an independent risk factor for conversion failure. A receiver operating characteristic curve constructed using duration of epidural labour analgesia showed that parturients with a duration of epidural labour analgesia ≥8 h, more frequently required a change of anaesthesia technique during conversion, and the relative risk of conversion failure was 1.54 (95% CI, 1.23-1.93; p < .01). CONCLUSION: Prolonged duration of epidural labour analgesia increases the possibility of having an invalid epidural catheter, resulting in an increased risk of conversion failure from epidural labour analgesia to epidural surgical anaesthesia. Further, this risk is higher when the time exceeds 8 h. KEY MESSAGESProlonged duration of epidural labour analgesia > 8 h is associated with conversion failure.If it is impossible to judge whether the conversion is successful immediately, spinal anaesthesia should be administered to minimise complications.

Evaluation and Recommendation of the 8th Edition of American Joint Committee on Cancer (AJCC) Staging System for Intrahepatic Cholangiocarcinoma (ICC) in 820 Patients from the Surveillance, Epidemiology, and End Results (SEER) Database.

BACKGROUND: The AJCC made four changes to T category in the 8th AJCC stage for ICC, but this is a topic of debate. METHODS: Data from 820 patients with ICC were extracted from the SEER database. Survival analysis of the 8th AJCC stage was examined. RESULTS: To verify the four T staging changes by survival analysis: prognosis of patients with tumor size > 5 cm was poorer than that with tumor size ≤ 5 cm (P < 0.05); in N0M0 cohort, there was no significant difference in survival between solitary tumor with vascular invasion and multiple tumors (P = 0.092), tumor perforating the visceral peritoneum with and without involving local extrahepatic structures by direct invasion (P = 0.470), and tumor with and without periductal invasion (PI) (P = 0.220). The prognosis of patients with ≥ 4 positive lymph nodes was relatively poor compared with 1-3 positive lymph nodes (P = 0.037) and similar to patients with stage IV (8th AJCC, P = 0.585). CONCLUSION: This study found that there was no significant difference in survival between tumor perforating the visceral peritoneum with and without involving local extrahepatic structures by direct invasion, whereas other T staging changes were effective. The inclusion of the number of positive lymph nodes in the 8th AJCC stage may improve prognostic discrimination in ICC patients.

First report of Cercospora cf. citrulina causing leaf spot of Ipomoea pes-caprae in China.

Ipomoea pes-caprae plays an important role in protecting the tropical and subtropical coastal beach of the world. In 2018, a leaf spot was observed on I. pes-caprae in Xisha islands of China, 13.2-25.8% of leaves were infected. The initial symptoms were small (1-3 mm diameter), single, circular, dark gray spots with a light-yellow center on the leaves. The lesions enlarged and were scattered or confluent, distinct and circular, subcircular or irregular, occasionally vein-limited, pale to dark gray-brown, with a narrow dark brown border surrounded by a diffuse yellow margin. Microscopic observations of the spots revealed that caespituli were dark brown and amphigenous, but abundant on the underside of the leaves. Mycelia were internal. Conidiophores were fasciculate, occasionally solitary, pale olivaceous-brown throughout, 0- to 3-septate, 27.9-115.8 (63.4±22.5) µm × 3.2-5.3 (4.3±0.87) µm (n=100). Conidial scars were conspicuously thickened. Conidia were solitary, hyaline, filiform, acicular to obclavate, straight to slightly curved, subacute to obtuse at the apex, truncate at the base, multi-septate, 21.0-125.5 (60.2±20.1) µm × 2.0-5.0 (3.8±0.83) µm (n=100). Single-conidium isolates were obtained from representative colonies grown on potato dextrose agar (PDA) incubated at 25℃ in the dark. The colonies grew slowly and were dense, white to gray and flat with aerial mycelium. Mycelia were initially white, and then became gray. Conidia were borne on the conidiophores directly. The pure isolate HTW-1 was selected for molecular identification and pathogenicity test, which were deposited in Microbiological Culture Collection Center of Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences. The internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1-alpha (tef1) and histone H3 (his3) genes were amplified with ITS1/ITS4, EF-1 / EF-2, and CYLH3F / CYLH3R primers, respectively (Groenewald et al. 2013). The obtained sequences of HTW-1 were all deposited in GenBank with accession numbers MT410467 for ITS, MT418903 for tef1 and MT418904 for his3. The ITS, tef1 and his3 genes all showed 100% similarity for ITS (JX143582), tef1 (JX143340) and his3 (JX142602) with C. cf. citrulina (MUCC 588; MAFF 239409) from I. pes-caprae in Japan. Based on the morphological characteristics and molecular identification, the pathogen was identified as Cercospora cf. citrulina (Groenewald et al. 2013). The pathogenicity test was conducted by spraying conidial suspension (1×104 conidia/mL) on wounded and unwounded leaves for seedling of I. pes-caprae in greenhouse and in sterile vitro condition. The conidial suspension was prepared using conidia from 30-day-old culture grown on PDA at 25℃ in the dark. Leaf surfaces of seedling in greenhouse were wounded by lightly rubbing with a steel sponge and detached leaf surfaces were wounded by sterile needles. the treatments were sprayed with conidial suspensions on wounded and unwounded leaf surfaces. The control was sprayed with sterile water. After eight days, the typical symptoms of spots which were small, single, circular and dark gray appeared on the inoculated wounded leaves, while the inoculated unwounded leaves and the control leaves were symptomless. The pathogen was only re-isolated from the inoculated wounded leaves. The pathogen may be infected by wound. A total of 20 Cercospora and related species was found on Ipomoea spp. (García et al. 1996). Cercospora cf. citrulina has been reported on I. pes-caprae in Japan, although it was unclear if it was a pathogen or saprophyte (Groenewald et al. 2013). To our knowledge, this is the first report of C. cf. citrulina causing leaf spot of I. pes-caprae in China. This disease could threat the cultivation of I. pes-caprae in China.

First report of Colletotrichum siamense causing anthracnose on Erythrina crista-galli in China.

Erythrina crista-galli L. (Fabaceae) is a popular ornamental plant in tropical and subtropical regions of South Asia. In October 2019, anthracnose-like lesions were observed on the leaves of E. crista-galli planted in Haikou, China. 5-30% of leaves were infected. At first, the circular spots of 1-2 mm in diameter were reddish-brown on the leaves, and then enlarged to circular, subcircular or irregular spots with reddish-brown center and surrounded by a diffuse yellow margin. Neighboring spots sometimes coalesced. Under continuously wet or humid conditions, the lesions expanded quickly, and became gray, subcircular or irregular spots covered by grayish-white mycelium and orange-pink conidial masses. Diseased leaves eventually fell off the trees. To identify the pathogen, diseased leaves were sampled from four gardens. Leaf tissues (5×5 mm) were cut from the margins of typical symptomatic lesions, surface-sterilized in 1% sodium hypochlorite for 1 min, plated on potato dextrose agar (PDA) medium, and incubated at 28.0±0.5℃ in the dark. Similar fungal colonies were obtained from all plated tissues after 3 days. The single-conidium colonies of all isolates were white to pale gray and cottony with visible orange conidial masses. Conidia were one-celled, aseptate, hyaline, straight, cylindrical to fusiform with obtuse ends, and ranged from 14.2-18.6 µm (16.4 µm)× 3.8-5.4 µm (4.7 µm) (n=100). After germination, conidia formed single, brown, oval or slightly irregular appressoria ranging from 8.0 to 11.8 µm (9.6 µm), and from 4.8 to 6.0 µm (5.4 µm). Sexual stage was absent. These characteristics of conidia and appressoria were matched with C. siamense belonging to the C. gloeosporioides complex (Prihastuti et al. 2009; Yang et al. 2009; Weir et al. 20012; Hu et al. 2015). To accurately identify the species, DNA was extracted from four purified isolates (JG-1, JG-3-1, SWS-1-3, SWS-2-1) （Fu et al. 2019）. The internal transcribed spacer of rDNA region (ITS), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), actin (ACT) and chitin synthase (CHS) genes were amplified and sequenced. The nucleotide sequences were all deposited in GenBank (ITS: MT229427-MT229430, GAPDH: MT250821-MT250824, CAL: MT258893-MT258896, ACT: MT258897-MT258900 and CHS: MT258901-MT258904). Multi-locus phylogenetic analyses (ITS, GAPDH, CAL, ACT and CHS) (Weir et al. 2012) showed that the four isolates were clustered with C. siamense, which was in accordance with BLAST results. Pathogenicity tests of the four isolates were repeated three times on detached leaves (Ji et al. 2019). The conidial suspension (1×106 conidia/mL) was prepared using the conidia from 10-day-old cultures grown on PDA. Two 20-µL drops of conidial suspension were inoculated on non-wounded young healthy leaves, and each isolate was inoculated on 10 leaves. Two 20-µL drops of sterile water were inoculated on non-wounded young healthy leaves as control. The samples were maintained in containers at a relative humidity of 90± 5 per cent inside and 28℃ with a 12-h photoperiod. Gray, subcircular spots similar to the field disease symptoms were observed on the all inoculated leaves after 7 days, whereas no visible symptoms appeared on the non-inoculated leaves. The pathogen was re-isolated from inoculated leaves thus fulfilling Koch's postulates. C. gloeosporioides has been previously reported as a pathogen causing leaf spot on Erythrina (E. indica var. picta, E. variegata var. orientalis) in Guam in 1983 and Brazil in 2012. (Russo et al. 1983; Oliveira et al. 2012). To our knowledge, this is the first report of C. siamense causing leaf spot of E. crista-galli in China.

Complete ureteral necrosis after injury sustained during lumbar disc surgery: A case report.

INTRODUCTION: Reports pertaining to ureteral injury sustained during lumbar disc surgery are rare; most ureteral injuries in this setting involve laceration or transection. PATIENT CONCERNS: We report a rare case of a 55-year-old man who presented with complete left ureteral necrosis 20 days after sustaining ureteral transection during lumbar disc surgery. DIAGNOSIS: The patient presented with seroperitoneum caused by left ureteral injury; post-operative histopathological examination of surgical specimen after discectomy had revealed ureter-like tissue. Exploratory laparoscopic surgery revealed necrosis of a long segment of ureter, which was not amenable to treatment with conventional methods. INTERVENTION: We used a spiral bladder muscle flap with vascular pedicles to repair the ureteral defect. OUTCOMES: Post-operative period was uneventful and the patient showed good recovery. CONCLUSION: Spiral bladder muscle flap with vascular pedicles may be used to repair extensive ureteric injury.

[Cloning, expression and immunity analysis of transketolase of Echinococcus granulosus].

OBJECTIVE: To obtain the prokaryotic expression of transketolase genes and analyze its value as a diagnostic antigen for echinococcosis. METHODS: TK gene was amplified by PCR and cloned into prokaryotic vector pMD19-EgTK, and then subcloned into the expression vector pET-28a. The target gene TK prokaryotic expression plasmid pET-28a was constructed and transferred into BL21. The purified protein was identified by SDS-PAGE and Western blotting. The blood samples of patients with cystic echinococcosis (CE group), alveolar echinococcosis (AE group) and healthy people (healthy group) were collected and detected by ELISA with the recombinant EgTK protein as a diagnostic antigen. RESULTS: The recombinant plasmid pET-28a (+)-EgTK was constructed successfully, and there was a band around 70 kDa by using Western blotting. ELISA showed that the difference among the 3 groups of sera reaction A450 was significantly different (F = 44.47, P < 0. 01), and the A450 values ofthe CE group (1.46±0.41) and AE group (1.28±0.29) were higher than that of the healthy group (0.66±0.23), but there was no significant difference between the former two. CONCLUSIONS: The recombinant EgTK protein is better to distinguish the echinococcosis group and healthy group, but it can't do a differential diagnosis between CE and AE cases.

Prospective, randomized, blinded, and placebo-controlled study of Cerebrolysin dose-response effects on long-term functional outcomes in a rat model of mild traumatic brain injury.

Using a prospective, randomized, blinded, placebo-controlled protocol, the authors demonstrated that Cerebrolysin at doses of 0.8-7.5 ml/kg, administered 4 hours after injury and then once daily for a total of 10 consecutive days, improves long-term functional outcomes in a rat model of mild closed head injury; a 2.5-ml/kg dose was identified as optimal. These findings suggest that Cerebrolysin has the potential to treat mild traumatic brain injury, the incidence of which is high without effective treatments.

Whole Exome Sequencing Identified a Novel IGFBP6 Variant in a Disc Degeneration Pedigree.

OBJECTIVE: To identify the likely causal mutation that results in disc degeneration in a pedigree with a high incidence of disc degeneration. MATERIALS AND METHODS: A large pedigree with a high incidence of disc degeneration was recruited for this study. Exome sequencing was completed on four family members with disc degeneration to screen for potential causal gene variants. Detected variants were filtered against the 1000 Genomes Project, the Short Genetic Variations database (dbSNP), and the Beijing Genomics Institute (BGI) in-house database. After removing synonymous variants, Sanger sequencing was used to verify the lack of the candidate single nucleotide polymorphism (SNP) in five healthy subjects of the study family. RESULTS: We identified a novel SNP variant, Chr12:g.53494591T>C. c.T430C (p.S144P) in the insulin-like growth factor binding protein-6 (IGFBP6) gene. This variant was shared by all four affected family members, but not by five unaffected members in the same pedigree. Furthermore, this variant was not detected in 200 unrelated healthy people. CONCLUSIONS: The c.T430C (p.S144P) variant of IGFBP6 was identified as the likely causal variant associated with increased risk of familial disc degeneration in the studied pedigree.

[Effect of calcitonin gene-related peptide on MC3T3-E1 osteoblast apoptosis and autophagy induced by serum starvation].

OBJECTIVE: To study the effect of calcitonin gene-related peptide (CGRP) on apoptosis and autophagy of mouse MC3T3-E1 osteoblast and their interaction and to further clarify protective mechanism of CGRP on osteoblasts. METHODS: MC3T3-E1 osteoblasts of mouse were cultured in vitro. Western blot and flow cytometry were used to detect expressions of microtubule-associated protein 1 light chain 3 (LC3) and P62 protein of MC3T3-E1 osteoblasts cultured with serum culture and serum-free (serum starvation) culture. Western blot was also used to detect expressions of LC3 and P62 protein of MC3T3-E1 osteoblast cultured at different concentrations (10⁻¹°, 10⁻9, 10⁻8, and 10⁻7 mol·L⁻¹) or without added CGRP. MC3T3-E1 osteoblasts were treated with 10⁻8 mol·L⁻¹ CGRP at different times (2, 6, 12, 24, 48, and 72 h), protein expression levels of LC3 were assessed by Western blot and flow cytometry, and changes in autophagosome in cells were detected by monodansylcadaverin staining. Autophagy inhibitor 3-methyladenine (3-MA) was used to pretreat MC3T3-E1 osteoblasts. Cells were then treated with or without CGRP for 24 h. Flow cytometry was used to detect apoptosis level. RESULTS: Under serum starvation conditions, LC3Ⅱ expression and apoptosis of osteoblasts increased compared with that of serum culture. Under 3-MA pretreatment and serum starvation conditions, LC3Ⅱ expression of osteoblasts increased compared with that of serum culture (P<0.01). Compared with serum culture, serum starvation culture with or without CGRP significantly increased expression level of LC3 and reduced expression level of P62. LC3Ⅱ/Ⅰ of osteoblasts was the highest under serum starvation and 10⁻8 mol·L⁻¹ CGRP conditions. Serum starvation and 10⁻8 mol·L⁻¹ CGRP culture inhibited apoptosis of osteoblasts and promoted synthesis of autophagosome. Apoptosis of osteoblasts increased after 3-MA pretreatment, and CGRP reversed inhibitory effects of 3-MA CGRP on apoptosis. CONCLUSIONS: CGRP can increase autophagy of MC3T3-E1 osteoblasts under serum starvation conditions. CGRP may also inhibit apoptosis of MC3T3-E1 osteoblasts by promoting autophagy.
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Two new compounds from Trollius chinensis Bunge.

Two new compounds, 2″-O-feruloylisoswertiajaponin (1) and (2E)-2-methyl-1-O-vaniloyl-4-ß-D-glucopyranoside-2-butene (2), along with one indole alkaloid and five known flavonoids, were isolated from the flowers of Trollius chinensis Bunge. Their structures were elucidated on the basis of spectroscopic evidence (UV, IR, HR-ESI-MS, NMR).

Targeting Triple Negative Breast Cancer with a Small-sized Paramagnetic Nanoparticle.

There is no available targeted therapy or imaging agent for triple negative breast cancer (TNBC). We developed a small-sized dendrimer-based nanoparticle containing a clinical relevant MRI contrast agent, GdDOTA and a NIR fluorescent dye, DL680. Systemic delivery of dual-modal nanoparticles led to accumulation of the agents in a flank mouse model of TNBC that were detected by both optical and MR imaging. In-vivo fluorescence images, as well as ex-vivo fluorescence images of individual organs, demonstrated that nanoparticles accumulated into tumor selectively. A dual modal strategy resulted in a selective delivery of a small-sized (GdDOTA)42-G4-DL680 dendrimeric agent to TNBC tumors, avoiding other major organs.

Relationship between Lung Volume and Pulmonary Function in Patients With Adolescent Idiopathic Scoliosis: Computed Tomographic-based 3-Dimensional Volumetric Reconstruction of Lung Parenchyma.

STUDY DESIGN: Retrospective. OBJECTIVES: We determined values for the volume of right lung (Vr), left lung (Vl), total lung volume (Vt), and left/right lung volume ratio (Vl/Vr), allowing comparison between those data measured and those of age-matched controls. To find whether lung volume correlates with preoperative pulmonary function. SUMMARY OF BACKGROUND DATA: To our knowledge, no study on relationship between computed tomographic (CT) scans determined lung volume and pulmonary function test (PFT) in scoliosis have been published. METHODS: All examinations with PFT (31 cases) were identified. Three-dimensional volumetric reconstruction of lung parenchyma was performed on existing preoperative CT scans for 26 idiopathic scoliosis patients. Vl, Vr, Vt, Vl/Vr, and absolute value of right volume minus left volume (|Vr-Vl|) were calculated and correlated with PFTs. To determine if significant difference of preoperative lung volume exists between idiopathic scoliosis patients and controls. Linear regression models, using 3-dimensional lung volume parameters as predictors for vital capacity (VC), forced vital capacity (FVC), and total lung capacity (TLC), were created. RESULTS: Vt was positively correlated with VC, FVC, forced expiratory volume in 1 second (FEV1), TLC, predicted value for FVC (FVC%), predicted value for FEV1 (FEV1%), predicted value for TLC (TLC%), and predicted value for maximal ventilator volume (MVV%) (P<0.05); |Vr-Vl| was not correlated with ventilation parameters (P>0.05); Diffusion parameters were not correlated with CT-reconstructed lung volume parameters (P>0.05); male and female adolescent idiopathic scoliosis patients had less Vt, Vr, and Vl compared with those of age-matched controls (P<0.05). CONCLUSIONS: Vt was positively correlated with VC, FVC, FEV1, TLC, FVC%, FEV1%, TLC%, and MVV%. Vt, Vr, and Vl of adolescent idiopathic scoliosis patients were less than those of age-matched controls.

[Calcitonin gene-related peptide-induced osteogenic differentiation of mouse bone marrow stromal cells through Hippo pathway in vitro].

OBJECTIVE: Previous studies have clarified that calcitonin gene-related peptide (CGRP) can promote the biologi- cal activity of osteoblasts. To further reveal the role of CGRP in bone repair, we studied its influence on osteogenic differentia- tion of mouse bone marrow stromal cells (BMSCs) and initially explored the effect of the Hippo signaling pathway with this process. METHODS: BMSCs were induced to osteogenic differentiate osteoblasts by different concentrations of CGRP for a screening of the optimal concentration. CGRP was added in BMSCs, then the activity of alkaline phosphatase (ALP) and the number of mineralized nodules were examined by specific ALP kits after 48 hours and alizarin red staining fluid after 7 days, respectively. The protein expression of p-Mst1/2 was measured by Western blot. Verteporfin was used to block the downstream Yap signaling. The mRNA expression of collagen type I (Col I) and runt-related transcription factor 2 (Runx2) were detected by reverse transcription-polymerase chain reaction. RESULTS: Compared to the blank group, different concentrations of CGRP (10⁻9, 10⁻8, 10⁻7 mol · L⁻¹), especially 10⁻8 mol · L⁻¹, significantly increased the ALP activity of BMSCs (P < 0.05). Alizarin red staining also showed more mineralized nodules in 10⁻8 mol · L⁻¹ group. The expression of p-Mst1/2 increased in the CGRP group (P < 0.05). Verteporfin treatment effectively decreased the mRNA expression of Runx2 and Col I (P < 0.05). CONCLUSION: The Hippo signaling pathway plays a role in CGRP-induced osteogenic differentiation in mouse BMSCs.

[Calcitonin gene-related peptide inhibits the expression of Nod-like receptor protein 3 to Dromote osteoblast differentiation in mouse osteoblasts in vitro].

OBJECTIVE: This study aims to investigate the regulatory effects of calcitonin gene-related peptide (CGRP) on Nod-like receptor protein 3 (NLRP3) and interleukin-1ß (IL-1ß) to promote osteoblast differentiation. METHODS: Different concentrations of CGRP (0, 10, 30, 100 ng · mL⁻¹) were added to mouse osteoblasts in vitro. The mRNA and protein expression levels of both NLRP3 and IL-1ß were examined using Real-time polymerase chain reaction and Western blot, respectively. Moreover, the concentrations of IL-1ß protein and intracellular reactive oxygen species (ROS) were detected using enzyme-linked immunosorbent assay and flow cytometry, respectively. The osteogenic differentiation of mouse osteoblasts was identified through alizarin red staining. RESULTS: The protein and mRNA expression levels of both NLRP3 and IL-1ß significantly decreased (P < 0.05) with increasing CGRP concentration. Moreover, the contents of intracellular ROS gradually decreased (P<0.05). The osteogenic differentiation of the osteoblasts was more enhanced in the group treated with 100 ng · mL⁻¹ CGRP than in the empty group (0 ng · mL⁻¹ CGRP). CONCLUSION: CGRP promotes osteoblast differentiation by inhibiting the expression of inflammatory factors.

Cerebrolysin dose-dependently improves neurological outcome in rats after acute stroke: A prospective, randomized, blinded, and placebo-controlled study.

BACKGROUND: Cerebrolysin is a mixture of neuropeptides and free amino acids that is clinically used for the treatment of stroke. To further standardize treatment schemes, we assessed the dose response of Cerebrolysin on sensorimotor outcome in a rat model of ischemic stroke. METHODS: This study was a prospective, blinded, placebo-controlled, preclinical experiment. Male and female Wistar rats, subjected to embolic middle cerebral artery occlusion, were randomly treated with Cerebrolysin doses of 0.8, 2.5, 5.0, 7.5 ml/kg or placebo, 4 h after middle cerebral artery occlusion for a total of 10 consecutive days. RESULTS: The primary outcome was neurologic improvement at day 28, lesion volume, mortality, and animal weight were secondary and safety outcomes, respectively. There was a significant (p < 0.001) dose effect of Cerebrolysin on neurological outcome. Cerebrolysin at a dose of ≥ 2.5 ml/kg significantly (p < 0.001) improved neurological outcome (Mean Estimate (95% CL): 0.8 ml/kg: 6.2 (-6.0/18.4), 2.5 ml/kg: -28.9 (-41.6/-16.2), 5.0 ml/kg: -33.4 (-45.0/-21.7), 7.5 ml/kg: -36.3 (-48.2/-24.4). Higher doses (≥ 2.5 ml/kg) resulted in better recovery; however, differences between effective doses were not significant. Treatment with 5 ml/kg reduced lesion volume (p = 0.016). No treatment gender interactions were found and there were no differences in death or weight loss. CONCLUSION: Collectively, these data on Cerebrolysin efficacy demonstrate the feasibility of a preclinical study setup following a randomized, placebo-controlled, and blinded design with a clinical relevant treatment scheme. Cerebrolysin at doses of ≥ 2.5 ml/kg improved functional outcome and at a dose of 5 ml/kg reduced infarct volume.

[Cloning, fusion expression and identification of thioredoxin encoding gene from Toxoplasma gondii].

OBJECTIVE: To clone and express the thioredoxin (Trx) from RH strain tachyzoites of Toxoplasma gondii, establish the prokaryotic expression vector and purify the recombinant protein, then produce the polyclonal anti-Trx antibody in rabbits. METHODS: Trx fragment was amplified by PCR and cloned into the pET-28a (+) vector, and the recombinant protein was induced with IPTG and purified by Ni-NTA affinity chromatography. The polyclonal antibody specificity was detected by Western blotting. RESULTS: The trx gene was amplified from T. gondii cDNA by PCR. The recombinant plasmid trx/pET-28a (+) was usefully constructed, and the recombinant TRX protein was expressed and purified. The TRX polyclonal antibody was also obtained. The specific band of TRX was detected by Western blotting. CONCLUSIONS: Western blotting can detect the specificity of polyclonal anti-Trx antibody, which will facilitate the biological functions of Trx.

[Protective effect of calcitonin gene-related peptide against oxidative damage in MC3T3-E1 osteoblasts].

OBJECTIVE: This study aimed to observe the protective effect of calcitonin gene-related peptide (CGRP), as well as its potential mechanism, against oxidative damage in MC3T3-E1 osteoblasts. METHODS: 1) MC3T3-E1 osteoblasts were treated with different hydrogen peroxide (H2O2) concentrations (10⁻¹, 10⁻², 10⁻³, 10⁻4, and 10⁻5 mol·L⁻¹) for 12, 24, 36, and 48 h to build an oxidative damage model, to determine cell proliferation activity in each group by using CCK-8 assay, and to determine the optimal modeling concentration. MC3T3-E1 osteoblasts were pretreated for 1 h with different CGRP concentrations (10⁻6, 10⁻7, 10⁻8, 10⁻9, and 10⁻¹° mol·L⁻¹) followed by treatment with H2O2 (10⁻4 mol·L⁻¹). After 12, 24, 36, and 48 h, the CGRP expression and activity of osteoblasts were detected using the CCK-8 method to determine the optimal CGRP concentration that provides the best protective effect against oxidative damage. 2) Superoxide dismutase (SOD) activity, reactive oxygen species (ROS) content, and the levels of the inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 of the groups treated with CGRP, H2O2, CGRP+H2O2 were determined. RESULTS: 1) Compared with the control group, treatment with 10⁻4 mol·L⁻¹ H2O2 significantly started to inhibite the proliferation of osteoblasts (P<0.01) in a dose- and time-dependent manner. Compared with 10⁻4 mol·L⁻¹ H2O2 group, pretreatment with 10⁻8 mol·L⁻¹ CGRP significantly increased the proliferation of osteoblasts (P<0.01). 2) Compared with H2O2 group, CGRP+H2O2 group significantly increased the SOD activity (P<0.01), ROS content significantly decreased (P<0.01), TNF-α, IL-1ß, and IL-6 secretion significantly decreased (P<0.05). CONCLUSIONS: H2O2 can cause oxidative damage to MC3T3-E1 osteoblasts, whereas CGRP exerts protective effect against oxidative damage in MC3T3-E1 osteoblasts.

A New Lignan Glucoside from Lagochilus ilicifolius.

BACKGROUND: The whole herb of Lagochilus ilicifolius has been used as a folk medicine for treating hemostatic, inflammation and ulcer in China. There were only limited reports on its chemical constituents, and no reports on its pharmacology study. OBJECTIVE: To isolate compounds from the whole herb of L. ilicifolius and evaluate their cytotoxic activity. MATERIALS AND METHODS: The column chromatographic techniques were used for separating the constituents of the n-butanol-soluble fraction of the 95% ethanol extract from the whole plant of L. ilicifolius. The structures of one new lignan and two known lignans were elucidated on the basis of spectroscopic analyses and comparison with literature data. The cytotoxic activities of these three lignans were evaluated using the MTT-assay against PC12 cell line derived from rat adrenal pheochromocytoma. RESULTS: The new lignan was identified as erythro-1-[(4-O-ß-D-glucopyranosyl-3-methoxyl)-phenyl]-2-[(5'-methoxyl)-pinoresinol]-propane-1,3-diol (1), and two known lignans were identified as tortoside C (2) and sisymbrifolin (3). The new lignan exhibited significant cytotoxic activity against PC12 cell line with IC50 value of 1.22 ± 0.03 µmol/L. CONCLUSIONS: A new lignan, erythro-1-[(4-O-ß-D-glucopyranosyl-3-methoxyl)-phenyl]-2-[(5'-methoxyl)-pinoresinol]-propane-1,3-diol and two known lignans were isolated from the whole herbs of L. ilicifolius. The two known lignans were reported for the first time in the genus Lagochilus. Three lignans were evaluated for in vitro cytotoxic activity. The new lignan showed relatively strong cytotoxicity against PC12 cell line, while sisymbrifolin and tortoside C exibited no cytotoxicity.