Layperson assessment of smile lines and upper lip combined images in smile esthetics.

STATEMENT OF PROBLEM: The esthetic assessment of smile lines by laypersons is a subject of ongoing debate. However, smile lines often appear with different types of upper lip curvature, which further complicates the esthetic assessment process, and studies on this combination are lacking. PURPOSE: The purpose of this clinical study was to investigate a layperson's esthetic perception of smile lines and upper lip combined images. MATERIAL AND METHODS: Twenty-six smile images resulting from combinations of 3 upper lip types, 4 anterior smile line types, and 3 posterior smile line types were generated by an image editing software program. Eighty-three laypersons (39 men and 44 women; 18 to 35 years of age) completed rating images using a visual analog scale. Unattractive smiles were designated to be those with scores <50 and attractive ones with scores ≥50. Data were analyzed using 1-way analysis of variance and Bonferroni post hoc tests (α=.05). RESULTS: High anterior smile line with gingival display >4 mm obtained significantly lower scores of <50 when combined with all upper lip curvatures (upward: 28.29 ±22.79, straight: 38.74 ±23.00, downward: 30.67 ±22.25, P<.01). High anterior smile lines with gingival display ≤4 mm combined with upward and straight upper lip curvature images obtained significantly higher scores, and all were ≥50 (upward: 63.24 ±22.22, straight: 61.40 ±21.58, P<.01). CONCLUSIONS: From a layperson's perspective, high anterior smile lines with gingival display >4 mm combined with any lip type were determined to be unattractive. If gingival display was ≤4 mm combined with both upward and straight lip types, the smile was assessed as attractive.

Quantitative evaluation of warm acupuncture-moxibustion in improvement of cartilage damage in rabbits with early knee osteoarthritis based on MR T2 mapping. / åŸºäºŽæ ¸ç£å ±æŒ¯T2 mappingå®šé‡è¯„ä»·æ¸©é’ˆç¸å¯¹æ—©æœŸå ”è†å ³èŠ‚éª¨å ³èŠ‚ç‚Žè½¯éª¨æŸä¼¤çš„ä¿®å¤ä½œç”¨.

OBJECTIVES: To observe the application value of MR T2 mapping for evaluating the effect of warm acupuncture-moxibustion on articular cartilage degeneration, and to observe the relationship between T2 value and expression of matrix metalloproteinases (MMP)-1 and MMP-13 of chondrocytes in rabbits with early knee osteoarthritis (KOA). METHODS: Thirty male New Zealand rabbits were randomly divided into blank control, KOA model and warm acupuncture-moxibustion groups, with 10 rabbits in each group. The early KOA model was established by right hind limb tubular plaster extension fixation method for 2 weeks. The rabbits of the warm acupuncture-moxibustion group received warm acupuncture-moxibustion stimulation at "Heding"(EX-LE2), "Neixiyan"(EX-LE4), "Waixiyan" (EX-LE5) and"Zusanli"(ST36) on the right hind limb for 15 min, once a day for 2 weeks. After intervention, MR T2 mapping of the right knee joint was performed in each group. The H.E. staining was used to evaluate the histopathological changes of cartilage, followed by giving a score according to the standards of Mankin scoring. The TUNEL method was used to analyze the apoptosis state of chondrocytes, and the positive expressions of MMP-1 and MMP-13 in the articular cartilage were detected by immunohistochemical staining. RESULTS: Compared with the blank control group, the Mankin score, chondrocyte apoptosis index, T2 value and the positive expressions of MMP-1 and MMP-13 in the cartilage tissue were significantly increased in the model group (P<0.01). Compared with the model group, the Mankin score, chondrocyte apoptosis index, T2 value and the positive expressions of MMP-1 and MMP-13 in the cartilage tissue were markedly decreased in the warm acupuncture-moxibustion group (P<0.01). The T2 value was positively correlated with the expression levels of MMP-1 and MMP-13 (P<0.01). H.E. staining showed disordered arrangement of chondrocytes and thinner cartilage layer in the model group, and a clear and relative ordered arrangement of chondrocyte in the warm acupuncture-moxibustion group. CONCLUSIONS: Warm acupuncture-moxibustion can reduce the T2 value of articular cartilage in early KOA rabbits, which is positively correlated with the decreased expression of MMP-1 and MMP-13 in the extracellular matrix of cartilage. The MR T2 mapping has certain value in evaluating the effect of warm acupuncture-moxibustion on KOA rabbits with early cartilage degeneration.

New Interpretation of Neonatal Outcomes by Phenotypically Classified Preterm Syndrome: A Retrospective Cohort Study.

OBJECTIVE: The global aim to lower preterm birth rates has been hampered by the insufficient and incomplete understanding of its etiology, classification, and diagnosis. This study was designed to evaluate the association of phenotypically classified preterm syndromes with neonatal outcomes; to what extent would these outcomes be modified after the obstetric interventions, including use of glucocorticoid, magnesium sulfate, and progesterone. METHODS: This was a retrospective cohort study conducted at Tongji Hospital (composed of Main Branch, Optical Valley Branch and Sino-French New City Branch) in Wuhan. A total of 900 pregnant women and 1064 neonates were retrospectively enrolled. The outcomes were the distribution of different phenotypes among parturition signs and pathway to delivery, the association of phenotypically classified clusters with short-term unfavorable neonatal outcomes, and to what extent these outcomes could be modified by obstetric interventions. RESULTS: Eight clusters were identified using two-step cluster analysis, including premature rupture of fetal membranes (PPROM) phenotype, abnormal amniotic fluid (AF) phenotype, placenta previa phenotype, mixed condition phenotype, fetal distress phenotype, preeclampsia-eclampsia & hemolysis, elevated liver enzymes, and low platelets syndrome (PE-E&HELLP) phenotype, multiple fetus phenotype, and no main condition phenotype. Except for no main condition phenotype, the other phenotypes were associated with one or more complications, which conforms to the clinical practice. Compared with no main condition phenotype, some phenotypes were significantly associated with short-term adverse neonatal outcomes. Abnormal AF phenotype, mixed condition phenotype, PE-E&HELLP phenotype, and multiple fetus phenotype were risk factors for neonatal small-for gestation age (SGA); placenta previa phenotype was not associated with adverse outcomes except low APGAR score being 0-7 at one min; mixed condition phenotype was associated with low APGAR scores, SGA, mechanical ventilation, and grade HI-W intraventricular hemorrhage (IVH); fetal distress phenotype was frequently associated with neonatal SGA and mechanical ventilation; PE-E&HELLP phenotype was correlated with low APGAR score being 0-7 at one min, SGA and neonatal intensive care unit (NICU) admission; multiple fetus phenotype was not a risk factor for the outcomes included except for SGA. Not all neonates benefited from obstetric interventions included in this study. CONCLUSION: Our research disclosed the independent risk of different preterm phenotypes for adverse pregnancy outcomes. This study is devoted to putting forward the paradigm of classifying preterm birth phenotypically, with the ultimate purpose of defining preterm phenotypes based on multi-center studies and diving into the underlying mechanisms.

Platelets reprogram monocyte functions by secreting MMP-9 to benefit postoperative outcomes following acute aortic dissection.

Platelets have a great ability to modulate immune responses. Monocyte-platelet aggregates (MPAs) are associated with the pathogenesis of cardiac disease. Notably, a low preoperative platelet count often indicates poor postoperative recovery following acute aortic dissection (AAD). The functions of platelets and MPAs in AAD, however, remain poorly understood. We found that, despite decreased platelet counts, platelets were also activated in AAD patients, with significant alterations in immune-modulating mediators. Of interest, monocytes in AAD patients had a suppressed immune status, which was correlated with poor outcomes following surgery. Interestingly, platelets preferentially aggregated with monocytes, and the levels of MPAs were related to recovery after surgical repair in AAD patients. Platelets restored suppressed monocyte functions in AAD patients by forming aggregates and partly by secreting matrix metalloproteinase-9 (MMP-9). Thus, the results point to a previously unknown mechanism for platelets involving monocyte reprogramming, which may improve postoperative outcomes following complex cardiovascular surgery.

A novel recombinant human microplasminogen induced complete posterior vitreous detachment without morphological change of retina in juvenile rabbits.

Vitreomacular traction syndrome results from persistent vitreoretinal adhesions in the setting of partial posterior vitreous detachment (PVD). Vitrectomy and reattachment of retina is an effective therapeutic approach. The adhesion between vitreous cortex and internal limiting membrane (ILM) of the retina is stronger in youth, which brings difficulties to induce PVD in vitrectomy. Several clinical investigations demonstrated that intravitreous injection of plasmin before vitrectomy could reduce the risk of detachment. In our study, a novel recombinant human microplasminogen (rhµPlg) was expressed by Pichia pastoris. Molecular docking showed that the binding of rhµPlg with tissue plasminogen activator (t-PA) was similar to plasminogen, suggesting rh µPlg could be activated by t-PA to generate microplasmin (µPlm). Moreover, rhµPlg had higher catalytic activity than plasminogen in amidolytic assays. Complete PVD was found at vitreous posterior pole of 125 µg rhµPlg-treated eyes without morphological change of retina in juvenile rabbits via intraocular injection. Our results demonstrate that rhµPlg has a potential value in the treatment of vitreoretinopathy.

[Warm acupuncture stimulation improves cartilage damage and motor function by regulating JAK2/STAT3 signaling pathway in rabbits with knee osteoarthritis].

OBJECTIVE: To observe the effect of warm acupuncture on the expression of Janus protein tyrosine kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway and inflammatory factors of articular cartilage in rabbits with knee osteoarthritis (KOA), so as to explore its underlying mechanisms in improving KOA. METHODS: New Zea-land rabbits were randomly divided into blank, model, warm acupuncture and medication groups (12 rabbits in each group). The KOA model was prepared by using the right hind limb tubular plaster extension fixation method. The rabbits in the warm acupuncture group received acupuncture of "Neixiyan"(EX-LE4),"Waixiyan"(ST35),"Heding"(EX-LE2) and "Zusanli"(ST36), followed by attaching an ignited moxa-stick segment to the acupuncture-handle. The treatment was conducted for 15 min, once a week for 4 weeks. The rabbits in the medication group received gavage of diclofenac sodium solution(0.35 mg/kg), once daily for 4 weeks. The dysfunction severity state of the rabbit's knee-joint was evaluated using Lequesne scale (0-3 points), and the histopathological changes of cartilage were observed under microscope after H.E. staining and the state of distribution of chondrocytes in different layers and the extracellular matrix was assessed using Mankin score (0-6 points). The contents of serum interleukin (IL)-6, tumor necrosis factor-α (TNF-α) and matrix metalloproteinase-9 (MMP-9) were measured by using ELISA, and the expression levels of p-JAK2/JAK2, p-STAT3/STAT3 and MMP-9 in knee cartilage tissue were detected using Western blot. RESULTS: Compared with the blank group, the Lequesne score, Mankin score, and the contents of serum IL-6, TNF-α and MMP-9, and the ratios of p-JAK2/JAK2 and p-STAT3/STAT3, and the expression level of MMP-9 protein in knee cartilage tissue were significantly increased in the model group (P<0.01). In comparison with the model group, the Lequesne score, Mankin score, contents of serum IL-6, TNF-α and MMP-9, and the ratios of p-JAK2/JAK2 and p-STAT3/STAT3, and the expression of MMP-9 protein in knee cartilage tissue were notably decreased in both the warm acupuncture and medication groups (P<0.01,P<0.05). The levels of Lequesne score, Mankin score, contents of serum IL-6, TNF-α and MMP-9, and the ratios of p-JAK2/JAK2 and p-STAT3/STAT3 in knee cartilage tissue were significantly lower in the warm acupuncture group than in the medication group (P<0.01, P<0.05). No significant difference was found between the warm acupuncture and medication groups in the expression of MMP-9 protein (P>0.05). Outcomes of H.E. showed injury of the perichondrium of knee joint, obvious reduction of the cartilage matrix staining, cystic changes, clustered and disordered arrangement and severe pyknosis and necrosis of the surface cells with reduction of number of cells and increase of vacuoles in the model group, which was milder in both warm acupuncture and medication groups. CONCLUSION: Warm acupuncture can improve motor function and reduce cartilage injury in KOA rabbits, which may be related to its functions in inhibiting the secretion of pro-inflammatory factors and regulating JAK2/STAT3 signaling and downregulating MMP-9 expression in the cartilage tissue.

[Study on mechanism of Rehmanniae Radix Praeparata for treatment of osteoarthritis based on network pharmacology and molecular docking].

The mechanism of Rehmanniae Radix Praeparata against osteoarthritis was investigated based on network pharmacology, molecular docking, and in vitro experiments in the present study. Osteoclast models were established via receptor activator of nuclear factor-κB ligand(RANKL) and macrophage colony-stimulating factor(M-CSF) inducing RAW264.7 cells. Further, the influence of Rehmanniae Radix Praeparata on the activity of tartrate-resistant acid phosphatase(TRAP) was evaluated and the efficacy of Rehmanniae Radix Praeparata in the treatment of osteoarthritis was verified. The active components of Rehmanniae Radix Praeparata were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and literature, and the potential targets of the components were collected from SwissTargetPrediction. Osteoarthritis disease targets were searched in Online Mendelian Inheritance in Man(OMIM), Therapeutic Target Database(TTD), GeneCards, and DisGeNET. The intersection targets of Rehmanniae Radix Praeparata and osteoarthritis were obtained by Venny platform. The protein-protein interaction(PPI) network was constructed by Cytoscape 3.8.2, and key targets were obtained based on topology algorithm. The Database for Annotation, Visualization and Integrated Discovery(DAVID) was used to perform Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis. Finally, the mRNA expression of the key targets was determined by RT-qPCR and the binding activity between the components and key targets was validated by molecular docking. The results showed that Rehmanniae Radix Prae-parata inhibited the TRAP activity, thus inhibiting bone resorption by osteoclasts and treating osteoarthritis. By network pharmacology, 14 active components of Rehmanniae Radix Praeparata and 126 intersection targets were obtained. The network pharmacology enrichment results revealed 432 biological processes and 139 signaling pathways. Key targets such as proto-oncogene tyrosine-protein kinase Src(SRC), signal transducer and activator of transcription 3(STAT3) and transcription factor p65(RELA) were obtained according to the degree in topological analysis. SRC was highly expressed in osteoclasts, which accelerated the development of osteoarthritis. Therefore, SRC was selected for subsequent verification, and Rehmanniae Radix Praeparata decreased the gene expression level of SRC. The molecular docking showed that acteoside, isoacteoside, raffinose had good bonding activity with SRC, suggesting that they might be the critical components in treating osteoarthritis. In conclusion, Rehmanniae Radix Praeparata can inhibit bone resorption by osteoclasts and balance the metabolism of articular cartilage and subchondral bone via acting on SRC, thus playing a therapeutic role in osteoarthritis. In addition, Rehmanniae Radix Praeparata may exert overall efficacy on osteoarthritis through other targets such as STAT3 and RELA, and other related pathways such as PI3 K-AKT and IL-17 signaling pathways.

[Anti-osteoarthritis components and mechanism of Fufang Duzhong Jiangu Granules].

Osteoarthritis is a common disease characterized by degenerative lesions of articular cartilage in the elderly.Fufang Duzhong Jiangu Granulues(FDJG), a classical prescription for the treatment of osteoarthritis, has the effects of nourishing liver and kidney, nourishing blood and sinew, and dredging collaterals and relieving pain.In this study, molecular simulation technology was combined with molecular biology methods to explore and verify the potential pharmacodynamic substances and molecular mechanism of FDJG in the treatment of osteoarthritis.Arachidonic acid(AA) metabolic pathway is a typical anti-inflammatory pathway, and secretory phospholipase A2 group ⅡA(sPLA2-ⅡA), 5-lipoxygenase(5-LOX), cyclooxygenase-2(COX-2), and leukotriene A4 hydrolase(LTA4 H) are the key targets of the pathway.Therefore, in this study, based on the pharmacophores and molecular docking models of the four key targets in AA pathway, a total of 1 522 chemical components in 12 medicinals of FDJG were virtually screened, followed by weighted analysis of the screening results in combination with the proportions of the medicinals in the prescription.The results showed that mainly 73 components in the preparation could act on the above four targets, suggesting they might be the potential anti-osteoarthritis components of FDJG.Considering the predicted effectiveness, availability, and compatibility of the medicinals, coniferyl ferulate, olivil, and baicalin were selected for further verification.Specifically, lipopolysaccharide(LPS)-induced RAW264.7 inflammatory cell model was used to verify the anti-inflammatory activity of the three components.The results showed that the three can effectively inhibit the release of NO, supporting the above selection.In addition, targets 5-LOX, COX-2, and LTA4 H had high activity, which suggested that they may be the key anti-osteoarthritis targets of FDJG.The comprehensive activity values of Eucommiae Cortex, Achyranthis Bidentatae Radix, Ginseng Radix et Rhizoma, Lycii Fructus, and Astragali Radix were much higher than that of other medicinals in the prescription, indicating that they may be the main effective medicinals in FDJG acting on the AA pathway.In this study, the potential anti-osteoarthritis components of FDJG were obtained.Moreover, it was clarified that the anti-osteoarthritis mechanism of FDJG was to act on LOX and COX pathway in AA metabolic pathway, which provided a reference for the study of pharmacodynamic substances and molecular mechanism of FDJG.

[Research status and strategy of quality control of medicinal and edible food].

As China is implementing "Healthy China" strategy, medicinal and edible food has attracted unprecedented attention due to the dual attributes of food and medicine. However, there is a lack of the quality control standard and the existing quality control research cannot fully reflect the dual attributes of medicinal and edible food, which consequently restrict the development of medicinal and edible food industry. This study reviewed the research status and proposed the strategy of quality control in line with the dual attribu-tes of medicinal and edible food, and clarified the research contents of quality control of medicinal and edible food of different types to provide references for the follow-up quality control of medicinal and edible food.

[Study on discovery of efficacy markers for Dachaihu Decoction and its action mechanism].

Dachaihu Decoction is a classical Chinese herbal prescription that is effective in harmonizing lesser yang and purging internal accumulated heat. At present, it has been widely used in clinical practice, and the resulting outcomes are satisfactory. However, its quality indicators and action mechanism are still not clear. Therefore, this paper explored the efficacy markers of Dachaihu Decoction and its action mechanism based on literature mining, molecular biology, and network pharmacology, so as to better control its quality and ensure its clinical efficacy. The efficacy markers of Dachaihu Decoction were predicted and analyzed according to the "five principles" for Q-markers of Chinese herbs. Then the anti-inflammatory activity of the efficacy markers of Dachaihu Decoction was evaluated with Griess reagent after the establishment of RAW264.7 cell inflammation model in vitro with lipopolysaccharide(LPS). The potential targets of efficacy markers were predicted by Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), ChEMBL, and SwissTargetPrediction, followed by the construction of the protein-protein interaction(PPI) network of the efficacy markers of Dachaihu Decoction. Topological, GO, and KEGG enrichment analysis was carried out to construct the "key target-signaling pathway-biological process" network, thus elucidating the action mechanism of the efficacy markers of Dachaihu Decoction. Saikosaponin B_2, baicalin, baicalein, wogonoside, neohesperidin, naringin, hesperidin, and paeoniflorin were considered as the potential efficacy markers of Dachaihu Decoction. The anti-inflammatory activity evaluation showed that the potential efficacy markers effectively inhibited the release of NO, exhibiting good anti-inflammatory activities. As demonstrated by network pharmacology, the efficacy markers of Dachaihu Decoction regulated the inflammatory response by acting on MAPK and NF-κB signaling pathways, the carbohydrate metabolism by HIF-1 and PI3 K-AKT signaling pathways, and the lipid metabolism by AMPK and PI3 K-AKT signaling pathways. This study discovered the efficacy markers of Dachaihu Decoction based on literature mining combined with molecular biological experiments and explored its action mechanism at the molecular level based on network pharmacology, which would provide reference for the quality control of Dachaihu Decoction and scientific basis for its clinical application.

Novel Fluorescence-Based High-Throughput FLIPR Assay Utilizing Membrane-Tethered Genetic Calcium Sensors to Identify T-Type Calcium Channel Modulators.

T-type voltage-gated Ca2+ channels have been implicated in many human disorders, and there has been increasing interest in developing highly selective and potent T-type Ca2+ channel modulators for potential clinical use. However, the unique biophysical properties of T-type Ca2+ channels are not conducive for developing high-throughput screening (HTS) assays to identify modulators, particularly potentiators. To illustrate, T-type Ca2+ channels are largely inactivated and unable to open to allow Ca2+ influx at -25 mV, the typical resting membrane potential of the cell lines commonly used in cellular screening assays. To address this issue, we developed cell lines that express Kir2.3 channels to hyperpolarize the membrane potential to -70 mV, thus allowing T-type channels to return to their resting state where they can be subsequently activated by membrane depolarization in the presence of extracellular KCl. Furthermore, to simplify the HTS assay and to reduce reagent cost, we stably expressed a membrane-tethered genetic calcium sensor, GCaMP6s-CAAX, that displays superior signal to the background compared to the untethered GCaMP6s or the synthetic Ca2+ sensor Fluo-4AM. Here, we describe a novel GCaMP6s-CAAX-based calcium assay utilizing a high-throughput fluorometric imaging plate reader (Molecular Devices, Sunnyvale, CA) format that can identify both activators and inhibitors of T-type Ca2+ channels. Lastly, we demonstrate the utility of this novel fluorescence-based assay to evaluate the activities of two distinct G-protein-coupled receptors, thus expanding the use of GCaMP6s-CAAX to a wide range of applications relevant for developing cellular assays in drug discovery.

In vitro fluorescence assay to measure GDP/GTP exchange of guanine nucleotide exchange factors of Rho family GTPases.

Guanine nucleotide exchange factors (GEFs) are enzymes that promote the activation of GTPases through GTP loading. Whole exome sequencing has identified rare variants in GEFs that are associated with disease, demonstrating that GEFs play critical roles in human development. However, the consequences of these rare variants can only be understood through measuring their effects on cellular activity. Here, we provide a detailed, user-friendly protocol for purification and fluorescence-based analysis of the two GEF domains within the protein, Trio. This analysis offers a straight-forward, quantitative tool to test the activity of GEF domains on their respective GTPases, as well as utilize high-throughput screening to identify regulators and inhibitors. This protocol can be adapted for characterization of other Rho family GEFs. Such analyses are crucial for the complete understanding of the roles of GEF genetic variants in human development and disease.

Warming moxibustion attenuates inflammation and cartilage degradation in experimental rabbit knee osteoarthriti.

OBJECTIVE: To explore the molecular mechanism of warming moxibustion (WM) in knee osteoarthritis (KOA). METHODS: The knee joints of 40 New Zealand rabbits were placed in a plaster cast in an extended position to establish a KOA model. The animals were randomly divided into four groups: the control group, model group, WM group, and diclofenac (DF) group. Hematoxylin-eosin staining and the modified Mankin score were applied to evaluate the histopathological changes. Chondrocyte apoptosis was determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Western blotting and real-time quantitative polymerase chain reaction were performed to measure the expression of interleukin-1α (IL-1ß), prostaglandin E receptor 3 (PTGER3), a disintegrin-like and metalloproteinase with thrombospondin type-1 motifs-5 (ADAMTS-5), matrix metalloproteinase-13 (MMP-13), and C-terminal telopeptides of collagen type II (CTX-II) in cartilage tissues of the different groups. The concentrations of IL-1ß, PTGER3, and CTX-II in serum were detected by the enzyme-linked immunosorbent assay. RESULTS: Rabbits with KOA in the WM and DF groups showed significantly reduced cartilage erosion and Mankin scores, compared with the untreated rabbits. The number of TUNEL-positive cells observed in the WM group was much fewer than that in the model group. The expression of PTGER3, MMP-13, CTX-II IL-1ß, and ADAMTS-5 in cartilage tissues was remarkably downregulated following therapy with WM and DF. Moreover, a marked reduction was observed in the serum levels of IL-1ß, PTGER3, and CTX-II in the WM and DF groups. CONCLUSION: WM exerts favorable therapeutic effects on articular injuries of KOA by regulating the expression of inflammatory and cartilage degradation-related cytokines.

Impact of the Glycemic Level on the Salivary Proteome of Middle-Aged and Elderly People With Type 2 Diabetes Mellitus: An Observational Study.

Type 2 diabetes mellitus (T2DM) is an increasing global public health concern, but its impact on the salivary proteome is still unclear. To evaluate the effect of glycemic levels in middle-aged and elderly individuals with T2DM on salivary proteomics, we compared the differences by liquid chromatography tandem mass spectrometry (LC-MS/MS). Unstimulated whole saliva samples from 8 T2DM patients with good glycemic control (G group, HbA1c <6.5%) and 16 patients with poor control (P group, HbA1c ≥6.5%) were analyzed by LC-MS/MS in the data-independent acquisition mode (Clinical register number: ChiCTR1900023582.). After functional annotation, cluster analysis and receiver operating characteristic (ROC) curve analysis were carried out to screen and evaluate candidate proteins. A total of 5,721 proteins were quantified, while 40 proteins differed significantly. In the P group, proteins involved in oxidative stress-related processes were upregulated, whereas proteins related to salivary secretion were downregulated. The combination of thioredoxin domain-containing protein 17, zymogen granule protein 16B, and FAM3 metabolism regulating signaling molecule D yielded an area under the curve of 0.917 which showed a robust ability to distinguish the P and G groups. In conclusion, poorly controlled hyperglycemia may affect salivary proteins through various pathways, including oxidative stress and glandular secretion. Furthermore, the differentially expressed proteins, especially the three proteins with the best differentiation, might serve as an anchor point for the further study of hyperglycemia and oral diseases.

[Identification of active components in Xuefu Zhuyu Decoction based on targets of blood-activating function].

As a classic prescription for promoting blood circulation to remove blood stasis, Xuefu Zhuyu Decoction(XFZYD) is widely used in clinical practice and has notable curative effect. Based on the key targets of activating blood circulation, this study identified the active components of XFZYD to reveal the material basis. The components of XFZYD were collected from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). The molecular docking models were built for the blood-activating targets obtained from the previous study with the components of XFZYD. The top five active components with measurability for each target were selected as the potential blood-activating components in the prescription. The efficacy of the prescription can embody key pharmacological and high-content components. In this study, anti-platelet aggregation activity was used to characterize the effect of activating blood, and the in vivo experiments were conducted to verify the accuracy of the active components. A total of 210 chemical components of XFZYD were screened out from TCMSP and docked with the key targets with the function of activating blood. Ligustrazine, acteoside, naringin, etc. were selected as the potential active components for activating blood in XFZYD. The anti-platelet aggregation activity of the combination of Chuanxiong Rhizoma, Rehmanniae Radix, Aurantii Fructus, Glycyrrhizae Radix et Rhizoma, and Carthami Flos was 9.82%±5.11%. Compared with that in the control group, the platelet aggregation induced by adenosine diphosphate(ADP) was significantly inhibited in the test group(P<0.01), which verified the accuracy of the active components. This study can guide the research on the material basis of XFZYD and provide insights into the development and utilization of the classical prescription.

[Material manufacturability classification in high shear wet granulation process of Chinese medicine].

The high shear wet granulation(HSWG) process of Chinese medicine has a complicated mechanism. There are many influencing factors that contribute to this process. In order to summarize the manufacturability of different kinds of materials in HSWG, this paper constructed a material library composed of 11 materials, including 4 Chinese medicine extracts and 7 pharmaceutical excipients. Each material was described by 22 physical parameters. Several binders were employed, and their density, viscosity and surface tension were characterized. Combining empirical constraints and the principle of randomization, 21 designed experiments and 8 verification experiments were arranged. The partial least squares(PLS) algorithm was used to establish a process model in prediction of the median granule size based on properties of raw materials and binders, and process parameters. The surface tension and density of binders, as well as the maximum pore saturation were identified as key variables. In the latent variable space of the HSWG process model, all materials could be divided into three categories, namely the Chinese medicine extracts, the diluents and the disintegrants. The granulation of Chinese medicine extracts required low viscosity and low amount of binder, and the resulted granule sizes were small. The diluent powders occupied a large physical space, and could be made into granules with different granule sizes by adjusting the properties of binders. The disintegrants tended to be made into large granules under the condition of aqueous binder. The combination use of material database and multivariate modeling method is conducive to innovate the knowledge discovery of the wet granulation process of Chinese medicine, and provides a basis for the formulation and process design based on material attributes.

[Potential therapeutic effect of Saussureae Involucratae Herba on breast cancer and its mechanism based on network pharmacology].

As one of the most commonly diagnosed cancers in the world, female breast cancer is induced by the high level of estrogen. Saussureae Involucratae Herba(SIH), a gynecological medicinal, regulates estrogen-induced diseases. However, the therapeutic effect of SIH on breast cancer has not been reported. Therefore, this study aims to explore the potential efficacy of SIH on breast cancer based on in vitro experiment and network pharmacology. The inhibitory effect of SIH water extract on proliferation and migration of breast cancer MDA-MB-231 cells was examined. The result demonstrated SIH water extract significantly suppressed the proliferation of breast cancer cells(IC_(50)=6.47 mg·mL~(-1)) and also restricted the migration. A total of 39 components of SIH were retrieved from traditional Chinese medicine database(TCMD) and 160 targets of SIH were screened by target fishing with the PharmaDB database. The Online Mendelian Inheritance in Man(OMIM) was used to establish a 1 001-targets data set of breast cancer. Based on the overlaps(45) of targets between SIH and breast cancer, a protein-protein interaction(PPI) network was built to analyze the interactions among these targets with STRING platform and Cytoscape. Finally, through topology and GO and KEGG analysis, 8 targets, 101 pathways and 85 biological processes were found to involve the treatment of breast cancer by SIH. SIH may exert the anti-breast cancer effect by regulating cell cycle, inhibiting proliferation, migration and adhesion of cancer cells, and modulating estrogen receptor. This study clarified the mechanism of SIH in treating breast cancer, which lays a foundation for the further development of SIH.

[Mechanism of Wuwei Ganlu in treatment of knee osteoarthritis:a study based on network pharmacology and molecular docking].

Wuwei Ganlu, a formula for medicated bath, consists of medicinal materials of Ephedra sinica, Platycladus orientalis, Myricaria squamosa, Artemisia carvifolia, and Rhododendron anthopogonoides, which is effective in inducing perspiration, resisting inflammation, relieving pain, regulating yellow water disease, and activating blood circulation. On this basis, a variety of formulas for Tibetan medicated bath have been derived for the treatment of diseases in internal organs, joints, nerves, etc. Modern studies have confirmed that Wuwei Ganlu has a good therapeutic efficacy on knee osteoarthritis(KOA). The present study explored the mechanism of Wuwei Ganlu in treating KOA based on network pharmacology and molecular docking. Firstly, the chemical components of Wuwei Ganlu were obtained through literature mining and database retrieval, and corresponding potential targets were predicted according to the BATMAN-TCM database. The protein-protein interaction(PPI) network was obtained after the potential targets were input into the STRING database. The network function modules were analyzed by the Molecular Complex Detection(MCODE) algorithm, and the functions of the modules were annotated to analyze the action mode of Wuwei Ganlu. Secondly, the related targets of KOA were collected through the DisGeNET database, and the overlapping targets were confirmed to analyze the mechanism of Wuwei Ganlu in treating KOA. Finally, the key targets were selected for molecular docking with the main components of Wuwei Ganlu to verify the component-target interaction. A total of 550 chemical components and 1 365 potential targets of Wuwei Ganlu were obtained. PPI analysis indicated that this formula could exert the effects of oxidation-reduction, inflammation resistance, bone absorption, bone mineralization, etc. Nineteen common targets were obtained from the intersection of potential targets of Wuwei Ganlu and KOA disease targets. It was found that the Wuwei Ganlu mainly acts on nuclear factor-κB(NF-κB), interleukin-1 beta(IL1ß), tumor necrosis factor(TNF), IL6, IL1 receptor antagonist(IL1 RN), and prostaglandin-endoperoxide synthase-2(PTGS2) to treat KOA. Among the 550 chemical components of Wuwei Ganlu, 252 potential active components were docked with TNF and 163 with PTGS2, indicating good binding of the components with potential key targets. The study preliminarily explored the mechanism of Wuwei Ganlu in treating KOA to provide a reference for the further development and utilization of Tibetan medicated bath that has been included in the UN Intangible Cultural Heritage.

[Analysis on targets of regulating Qi and activating blood based on activity spectrum of targets].

The traditional Chinese medicines(TCM) for activating blood circulation and the TCM for regulating Qi are often used in combination in clinical practice. However, their mechanisms are still unclear. The activity spectrum of targets can fuse the active components, targets and intensity of action, which provides support for the discussion of efficacy targets. The chemical components of common TCM sets for activating blood circulation and regulating Qi, as well as the negative sets not for activating blood circulation and re-gulating Qi were obtained from the database of TCM. By the similarity analysis of chemical components in TCM for activating blood circulation and DrugBank database, the predicted targets of chemical components in TCM for activating blood circulation were obtained, and the similarity value of the two was taken as the activity value of the active components and predicted targets. Then, the component-target activity value was weighted. The activity values of herb acting on the same target were fused to construct activity spectra of targets of the herbs for activating blood circulation, herbs for regulating Qi and negative herbs. The targets whose activity values of activating blood circulation and regulating Qi were higher than those of negative herbs were selected as potential targets of efficacy. Protein-protein interaction networks were constructed for topological, GO and KEGG enrichment analysis to determine the key targets of efficacy of activating blood circulation and regulating Qi. The component-target activity information collected from DrugBank database contained 4 499 compounds, 627 targets and 11 295 action relationships. The activating blood function protein-protein interaction network contained 206 nodes and 1 728 edges, while the regulating Qi function protein-protein interaction network contained 230 nodes and 986 edges. The enrichment analysis of topology, GO and KEGG showed that TCM for activating blood circulation mainly exerted its anti-inflammatory, neuroprotective and angiogenic effects on signaling cascade pathway mediated by VEGF/VEGFR2, ERK signaling pathway, calcium signaling pathway and PI3 K-AKT signaling pathway, and the key targets included mitogen activated protein kinases 3(MAPK3), proto-oncogene tyrosine-protein kinase Src(SRC), mitogen activated protein kinases 1(MAPK1), epidermal growth factor receptor(EGFR), phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform(PIK3 CA), peroxisome proliferators-activated receptor gamma(PPARG), nitric oxide synthase 3(NOS3), prostaglandin G/H synthetase 2(PTGS2), matrix metalloproteinase-9(MMP9), and vascular endothelial growth factor A(VEGFA). TCM for regulating Qi mainly exerted anti-inflammatory and neuroprotective effects by acting on MAPK signaling pathway and PI3 K-AKT signaling pathway, and the key targets included mitogen activated protein kinases 8(MAPK8), SRC, mitogen activated protein kinases 14(MAPK14), and RAC-alpha serine/threonine-protein kinase(AKT1), mitogen activated protein kinases 3(MAPK3). Based on the activity spectrum of targets, the targets of the TCM for activating blood and the targets of the TCM for regulating Qi were analyzed to provide reference for the study of efficacy targets of TCM, and also provide some scientific basis for clinical application.

VEGF121 Mediates Post-Hypoxia Cardioprotective Effects Via CaSR and Mitochondria-Dependent Protease Pathway. / VEGF121 como Mediador de Efeitos Cardioprotetores Pós-Hipóxia via CaSR e via da Protease Dependente de Mitocôndria.

BACKGROUND: Cardiovascular disease is the major cause of death worldwide. Hypoxia-mediated apoptosis in cardiomyocytes is a major cause of cardiovascular disorders. Treatment with vascular endothelial growth factor (VEGF) protein has been tested but operational difficulties have limited its use. However, with the advancements of gene therapy, interest has risen in VEGF-based gene therapy in cardiovascular disorders. However, the precise mechanism by which VEGF replenishment rescues post-hypoxia damage in cardiomyocytes is not known. OBJECTIVES: To investigate the effect of post-hypoxia VEGF121 expression using neonatal rat cardiomyocytes. METHODS: Cardiomyocytes isolated from neonatal rats were used to establish an in vitro model of hypoxia-induced cardiac injury. The effect of VEGF overexpression, alone or in combination with small-molecule inhibitors targeting calcium channel, calcium sensitive receptors (CaSR), and calpain on cell growth and proliferation on hypoxia-induced cardiomyocyte injury were determined using an MTT assay, TUNEL staining, Annexin V/PI staining, lactate dehydrogenase and caspase activity. For statistical analysis, a value of P<0.05 was considered to be significant. RESULTS: The effect of VEGF121 was found to be mediated by CaSR and calpain but was not dependent on calcium channels. CONCLUSIONS: Our findings, even though using an in vitro setting, lay the foundation for future validation and pre-clinical testing of VEGF-based gene therapy in cardiovascular diseases.

FUNDAMENTO: A doença cardiovascular é a principal causa de morte em todo o mundo. A apoptose mediada por hipóxia em cardiomiócitos é uma das principais causas de distúrbios cardiovasculares. O tratamento com a proteína do fator de crescimento endotelial vascular (VEGF, do inglês vascular endothelial growth factor) foi testado, mas as dificuldades operacionais limitaram seu uso. Entretanto, com os avanços da terapia gênica, aumentou o interesse na terapia gênica baseada no VEGF em doenças cardiovasculares. No entanto, o mecanismo preciso pelo qual a reposição de VEGF resgata os danos pós-hipóxia em cardiomiócitos não é conhecido. OBJETIVOS: Investigar o efeito da expressão de VEGF121 pós-hipóxia utilizando cardiomiócitos de ratos neonatos. MÉTODOS: Cardiomiócitos isolados de ratos neonatos foram utilizados para estabelecer um modelo in vitro de lesão cardíaca induzida por hipóxia. O efeito da superexpressão de VEGF, isolado ou em conjunto com inibidores de moléculas pequenas que têm como alvo os canais de cálcio, receptores sensíveis ao cálcio (CaSR, do inglês calcium-sensitive receptors) e calpaína, no crescimento e proliferação celular em lesão de cardiomiócitos induzidos por hipóxia, foram determinados com ensaio de MTT, coloração TUNEL, coloração com Anexina V/PI, lactato desidrogenase e atividade da caspase. Para análise estatística, um valor de p<0,05 foi considerado significativo. RESULTADOS: Verificou-se que o efeito do VEGF121 foi mediado por CaSR e calpaína, mas não foi dependente dos canais de cálcio. CONCLUSÕES: Nossos resultados, mesmo em um ambiente in vitro, estabelecem as bases para uma validação futura e testes pré-clínicos da terapia gênica baseada em VEGF em doenças cardiovasculares.