The Extracts of Dendrobium Alleviate Dry Eye Disease in Rat Model by Regulating Aquaporin Expression and MAPKs/NF-κB Signalling.

Dry eye is one of the most common ocular surface diseases caused by tear film instability and ocular surface damage due to an abnormal quality or quantity of tears. Inflammatory factors can initiate relevant transduction signalling pathways and trigger the inflammatory cascade response, resulting in ocular surface inflammation. It has been shown that the active ingredients in Dendrobium, such as polysaccharides, alkaloids and phenols, have anti-inflammatory, anti-tumour and immunity-boosting effects, and Dendrobium officinale extract can improve glandular secretion function, increase salivary secretion and increase the expression level of water channel protein in salivary glands in patients with dry eye syndromes. We investigated the in vitro cytoprotective effect of Dendrobium extracts in sodium chloride induced hyperosmotic conditions in human cornea keratocytes (HKs). Results showed that Dendrobium officinale Kimura et Migo water extract (DOW) and Dendrobium loddigesii Rolfe water extract (DLW) could upregulate the expression of aquaporins (AQP)5 protein, thus exerting a repairing effect by promoting cell migration. Furthermore, oral administration of DOW and DLW enhanced tear production in rats and exerted a protective effect on ocular surface damage. DOW and DLW could upregulate the expression of AQP5 and mucin (muc)5ac proteins in the lacrimal gland and reduce the inflammatory response. DOW and DLW inhibited the activation of the corresponding mitogen-activated protein kinases (MAPK) and NF-KB pathway, thereby playing a role in improving dry eye symptoms. This study provides a new perspective on dry eye treatment, and DOW and DLW may be potential therapeutic agents for dry eye.

Oxybutynin nanosuspension gel for enhanced transdermal treatment for overactive bladder syndrome.

Oxybutynin (OXY) is the most common drug to treat overactive bladder (OAB) syndrome. Transdermal administration is a more ideal route replacing oral administration to resolve problems of low bioavailability and severe side effects. However, commercial transdermal products of OXY frequently cause skin irritation and low permeation efficiency arising discontinued medication. Here, oxybutynin nanosuspension (OXY-NS) and its gel preparation (OXY-NG) were constructed to resolve these issues. In vitro permeation test and in vivo pharmacokinetics study confirmed that OXY-NG significantly enhanced the transdermal permeation of OXY, about 4-fold and 3-fold higher than oxybutynin coarse suspension (OXY-CG), respectively, and in vitro retention test certified that OXY-NG increased OXY concentration especially in viable epidermis (VE) and Dermis (about 3 times that of OXY-CG), consequently improving the bioavailability. Skin irritation assay demonstrated that OXY-NG would not trigger skin adverse effects. In addition, selectively blocking hair follicles test evidenced that hair follicles pathway played an important role in OXY-NS transdermal delivery. In general, by virtue of excellent drug loading, low toxicity and ease of scale-up, OXY-NG is a promising strategy to ameliorate skin permeation of insoluble OXY for better transdermal treatment for OAB, hence increasing its bioavailability, reducing adverse effects, and achieving good patient compliance.

Influence of cryopreservation methods of ex vivo rat and pig skin on the results of in vitro permeation test.

In vitro permeation test (IVPT) is a frequently used method for in vitro assessment of topical preparations and transdermal drug delivery systems. However, the storage of ex vivo skin for IVPT remains a challenge. Here, two cryopreservation media were chosen to preserve rat and pig skin at -20 °C and -80 °C for further IVPT, namely, 10 % DMSO and 10 % GLY. The skin viability test confirmed that the skin protective capacity of 10 % DMSO and 10 % GLY was almost equal. The results of skin viability and IVPT showed that the skin viability and permeability of rat skin in 10 %DMSO or 10 % GLY were maintained for at least 7 and 30 days at -20 °C and -80 °C compared to fresh skin, respectively; in contrast, those of porcine skin were just maintained for <7 days at -20 °C and -80 °C. These results indicated that ex vivo skin for IVPT preserved at -80 °C in 10 % DMSO or 10 % GLY was optimal. Furthermore, skin permeability was independent of skin barrier integrity. Our study provides reference conditions for preserving IVPT skin, and skin viability can be a potential indicator of IVPT skin.

Network pharmacology and metabolomics elucidate the underlying mechanisms of Venenum Bufonis in the treatment of colorectal cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: The present study aims to evaluate the efficacy of Venenum Bufonis (VBF), a traditional Chinese medicine derived from the dried secretions of the Chinese toad, in treating colorectal cancer (CRC). The comprehensive roles of VBF in CRC through systems biology and metabolomics approaches have been rarely investigated. AIMS OF THE STUDY: The study sought to uncover the potential underlying mechanisms of VBF's anti-cancer effects by investigating the impact of VBF on cellular metabolic balance. MATERIALS AND METHODS: An integrative approach combining biological network analysis, molecular docking and multi-dose metabolomics was used to predict the effects and mechanisms of VBF in CRC treatment. The prediction was verified by cell viability assay, EdU assay and flow cytometry. RESULTS: The results of the study indicate that VBF presents anti-CRC effects and impacts cellular metabolic balance through its impact on cell cycle-regulating proteins, such as MTOR, CDK1, and TOP2A. The results of the multi-dose metabolomics analysis suggest a dose-dependent reduction of metabolites related to DNA synthesis after VBF treatment, while the EdU and flow cytometry results indicate that VBF inhibits cell proliferation and arrests the cell cycle at the S and G2/M phases. CONCLUSIONS: These findings suggest that VBF disrupts purine and pyrimidine pathways in CRC cancer cells, leading to cell cycle arrest. This proposed workflow integrating molecular docking, multi-dose metabolomics, and biological validation, which contented EdU assay, cell cycle assay, provides a valuable framework for future similar studies.

A Q-marker screening strategy based on ADME studies and systems biology for Chinese herbal medicine, taking Qianghuo Shengshi decoction in treating rheumatoid arthritis as an example.

Chinese herbal medicine (CHM) exhibits a broad spectrum of clinical applications and demonstrates favorable therapeutic efficacy. Nonetheless, elucidating the underlying mechanism of action (MOA) of CHM in disease treatment remains a formidable task due to its inherent characteristics of multi-level, multi-linked, and multi-dimensional non-linear synergistic actions. In recent years, the concept of a Quality marker (Q-marker) proposed by Liu et al. has significantly contributed to the monitoring and evaluation of CHM products, thereby fostering the advancement of CHM research. Within this study, a Q-marker screening strategy for CHM formulas has been introduced, particularly emphasising efficacy and biological activities, integrating absorption, distribution, metabolism, and excretion (ADME) studies, systems biology, and experimental verification. As an illustrative case, the Q-marker screening of Qianghuo Shengshi decoction (QHSSD) for treating rheumatoid arthritis (RA) has been conducted. Consequently, from a pool of 159 compounds within QHSSD, five Q-markers exhibiting significant in vitro anti-inflammatory effects have been identified. These Q-markers encompass notopterol, isoliquiritin, imperatorin, cimifugin, and glycyrrhizic acid. Furthermore, by employing an integrated analysis of network pharmacology and metabolomics, several instructive insights into pharmacological mechanisms have been gleaned. This includes the identification of key targets and pathways through which QHSSD exerts its crucial roles in the treatment of RA. Notably, the inhibitory effect of QHSSD on AKT1 and MAPK3 activation has been validated through western blot analysis, underscoring its potential to mitigate RA-related inflammatory responses. In summary, this research demonstrates the proposed strategy's feasibility and provides a practical reference model for the systematic investigation of CHM formulas.

Single-nucleotide polymorphism array coupled with multiple displacement amplification: accuracy and spatial resolution for analysis of chromosome copy numbers in few cells.

When coupled with multiple displacement amplification (MDA), microarray-based comparative genomic intensity allows detection of chromosome copy number aberrations even in single or few cells, but the actual performance of the system and their influencing factors have not been well defined. Here, using single-nucleotide polymorphism (SNP) array, we analyzed copy number profiles from DNA amplified by MDA in 1-10 cells and estimated the accuracy and spatial resolution of the analysis. Based on the concordance of SNP copy numbers for DNA with and without MDA, the accuracy of the system can be significantly enhanced by using MDA-amplified DNA as reference and also by increasing the cell numbers. Analyses under different smoothing treatments revealed a practical resolution of 2 Mb for 10 cells and 10 Mb for a single cell. When both cells with known chromosomal duplication and deletion were analyzed, this platform detected a copy number "loss" more accurately than a "gain" (P < 0.01), particularly in single-cell MDA products. Together, we demonstrated that SNP array coupled with MDA is reliable and efficient for detection of copy number aberrations in a small number of cells, and its accuracy and resolution can both be significantly enhanced with increasing the number of cells as MDA template.

The Modulatory Effects and Targets Prediction of Herbal Medicines or Phytochemicals on Cancer Immunosurveillance.

Cancer is a main life-threatening disease worldwide. Due to the adverse effects of conventional chemotherapies and radiotherapies, immunotherapy has emerged as a potent strategy to treat cancer. In cancer immunotherapy, cancer immune surveillance plays a crucial role in the cancer process, which contains various effector cells from innate and adaptive immunity. This review summarized the functions of innate and adaptive immune cells in cancer immunosurveillance and their main reported targets. Moreover, the potential targets about the modulatory effects of cancer immunosurveillance were predicted using network-based target analysis, with total predicted pathways not only reporting previously reported pathways, but also putative signaling pathways pending for investigation. In addition, the potential use of herbal medicines and their phytochemicals in the modulation of cancer immunosurveillance were also discussed. Taken together, this review paper aims to provide scientific insight into further drug development, particularly herbs, phytochemicals, and TCM formulae, in the modulatory effects of cancer immunosurveillance.

Meteorin-ß/Meteorin like/IL-41 attenuates airway inflammation in house dust mite-induced allergic asthma.

We sought to examine the regulatory effect of Meteorin-ß (Metrnß)/Meteorin like (Metrnl)/IL-41 on lung inflammation in allergic asthma. We found that Metrnß was elevated significantly in asthmatic patients and in mice with allergic asthma induced by house dust mite (HDM) extract. Upon exposure to HDM, Metrnß was secreted predominantly by airway epithelial cells and inflammatory cells, including macrophages and eosinophils. The increased Metrnß effectively blocked the development of airway hyperreactivity (AHR) and decreased inflammatory cell airway infiltration and type 2 cytokine production, which was associated with downregulated DC-mediated adaptive immune responses. Moreover, Metrnß impaired the maturation and function of bone marrow-derived dendritic cells in vitro. Asthmatic mice adoptively transferred with dendritic cells isolated from Metrnß-treated allergic mice displayed decreased AHR, airway inflammation, and lung injury. Metrnß also displayed anti-inflammatory properties in immunodeficient SCID mice with allergic asthma and in in vitro 3D ALI airway models. Moreover, blockade of Metrnß by anti-Metrnß antibody treatment promoted the development of allergic asthma. These results revealed the unappreciated protective roles of Metrnß in alleviating DC-mediated Th2 inflammation in allergic asthma, providing the novel treatment strategy of therapeutic targeting of Metrnß in allergic asthma.

Emerging Roles on Immunological Effect of Indoleamine 2,3-Dioxygenase in Liver Injuries.

Indoleamine 2,3-dioxygenase (IDO) is one of the initial rate-limiting enzymes of the kynurenine pathway (KP), which causes immune suppression and induction of T cell anergy. It is associated with the imbalance of immune homeostasis in numerous diseases including cancer, chronic viral infection, allergy, and autoimmune diseases. Recently, IDO has extended its role to liver field. In this review, we summarize the dysregulation and potentials of IDO in the emerging field of liver injuries, as well as current challenges for IDO targets. In particular, we discuss unexpected conclusions against previous work published. IDO is induced by pro-inflammatory cytokines in liver dysfunction and exerts an immunosuppressive effect, whereas the improvement of liver injury may require consideration of multiple factors besides IDO.

Current Advances in Mechanisms and Treatment of Dry Eye Disease: Toward Anti-inflammatory and Immunomodulatory Therapy and Traditional Chinese Medicine.

Dry eye is currently one of the most common ocular surface disease. It can lead to ocular discomfort and even cause visual impairment, which greatly affects the work and quality of life of patients. With the increasing incidence of dry eye disease (DED) in recent years, the disease is receiving more and more attention, and has become one of the hot research fields in ophthalmology research. Recently, with the in-depth research on the etiology, pathogenesis and treatment of DED, it has been shown that defects in immune regulation is one of the main pathological mechanisms of DED. Since the non-specific and specific immune response of the ocular surface are jointly regulated, a variety of immune cells and inflammatory factors are involved in the development of DED. The conventional treatment of DED is the application of artificial tears for lubricating the ocular surface. However, for moderate-to-severe DED, treatment with anti-inflammatory drugs is necessary. In this review, the immunomodulatory mechanisms of DED and the latest research progress of its related treatments including Chinese medicine will be discussed.

[Evaluation of the fidelity of multiple displacement amplification from small number of cells].

OBJECTIVE: To evaluate the fidelity of multiple displacement amplification (MDA) from small number of cells (1-10 cells) by 10K 2.0 SNP mapping array. METHODS: A fibroblast cell line (Tri-18; GM02732, 47, XY, +18) was used as the template, and 6 groups were set up in the study. Groups A and B were positive and negative control, respectively; groups C-F were experimental groups involving the MDA products from 1, 2, 5 and 10 cells respectively. In combination of single nucleotide polymorphism (SNP) array, the product of each group was assessed based on the genome coverage, loss of heterozygosity (LOH) rate and allele dropout (ADO) rate. RESULTS: The nonspecific product of negative control presented an average call rate of 3.2%. The genome coverage of the MDA product increased from 86.4% to 96.4% with the increasing number of template from 1 to 10 cells, while the LOH rate and ADO rate decreased significantly (P<0.05). CONCLUSION: MDA is a highly efficient and reliable method for whole genome amplification. The fidelity of MDA will be improved significantly with the increasing number of template cells. 10K 2.0 SNP mapping array is a quick, accurate and comprehensive method to evaluate the fidelity of amplified DNA products, but the ADO SNPs should be distinguished from those of preferential amplification from the LOH loci to avoid errors.

Evaluation of genome coverage and fidelity of multiple displacement amplification from single cells by SNP array.

The scarce amount of DNA contained in a single cell is a limiting factor for clinical application of preimplantation genetic diagnosis mainly due to the risk of misdiagnosis caused by allele dropout and the difficulty in obtaining copy number variations in all 23 pairs of chromosomes. Multiple displacement amplification (MDA) has been reported to generate large quantity of products from small amount of templates. Here, we evaluated the fidelity of whole-genome amplification MDA from single or a few cells and determined the accuracy of chromosome copy number assessment on these MDA products using an Affymetrix 10K 2.0 SNP Mapping Array. An average coverage rate (86.2%) from single cells was obtained and the rates increased significantly when five or more cells were used as templates. Higher concordance for chromosome copy number from single cells could be achieved when the MDA amplified product was used as reference (93.1%) than when gDNA used as reference (82.8%). The present study indicates that satisfactory genome coverage can be obtained from single-cell MDA which may be used for studies where only a minute amount of genetic materials is available. Clinically, MDA coupled with SNP mapping array may provide a reliable and accurate method for chromosome copy number analysis and most likely for the detection of single-gene disorders as well.

Biochemical and computational evaluation of Triptolide-induced cytotoxicity against NSCLC.

Triptolide is the major bioactive component isolated from the Chinese Medicinal plant Tripterygium wilfordii. Despite the growing interest and the plethora of reports discussing the pharmacological activity of this diterpenoid, no clear consensus regarding its cellular targets and full mechanism of action has been reached. In the present work, a combined in vitro and in silico approach was used to evaluate the biological activity of Triptolide on Non-small cell lung cancer (NSCLC). In vitro, Triptolide treatment induced apoptosis in NSCLC cell lines and down-regulated the phosphorylation of AKT, mTOR, and p70S6K. Triptolide also impacted cellular glycolysis as well as the antioxidant response through the impairment of glucose utilization, HKII, glutathione, and NRF2 levels. Molecular docking results examined the possible interactions between Triptolide and AKT and predicted an allosteric binding to AKT-1 structure. Molecular dynamics simulations were further used to evaluate the stability of the complex formed by Triptolide's best conformer and AKT. These findings provide an insightful approach to the anticancer effect of Triptolide against NSCLC and highlight a possible new role for AKT/mTOR HKII inhibition.

Non-invasive pre-implantation aneuploidy screening and diagnosis of beta thalassemia IVSII654 mutation using spent embryo culture medium.

BACKGROUND: Cell-free nuclear DNA has been isolated from spent embryo culture medium. Whether this small amount of DNA can be amplified at the whole genome level and the concordance rate of karyotypes and specific alleles between biopsied cells and media has not been evaluated. METHODS: Seven couples were recruited, 88 donated embryos and their corresponding media were collected for whole genome amplification (WGA). The efficiency of WGA, the concordance of chromosome status, and the HBB gene IVSII654 allele between biopsied cells and media were investigated. RESULTS: After WGA, the DNA detection rate was 90.90% with a mean concentration of 26.15 ng/µl. The full chromosome concordance rate between biopsied cells and medium was 64.52%, and it increased to 90.00% for diploid blastocyst samples. Analysis of the mutated IVSII654 locus and SNP linkage verified that the DNA present in the medium originated from embryonic cells. CONCLUSION: We confirmed that nuclear DNA is present in spent culture medium and that the majority of this DNA can be amplified for subsequent analysis. Our results showed that non-invasive embryo genetic testing at the chromosomal-level using medium can concordant to the biopsied cells, but it needs further optimized before use in clinical applications. KEY MESSAGES The aggressive biopsy step during PGD/PGS procedure would have a negative effect on the future development of the embryo. Cell-free nuclear DNA has been observed in spent embryo culture medium, which holds promise for the development of non-invasive PGD/PGS approaches. The presence of DNA in medium, its efficiency for WGA, and the concordance between chromosome status and the HBB gene IVSII654 allele as diagnosed from biopsied cells or medium were investigated. Non-invasive embryo genetic testing at the chromosomal-level and allele site using medium can concordant to the biopsied cells, but it needs further optimized before use in clinical applications.

ß-Elemene attenuates atherosclerosis in apolipoprotein E-deficient mice via restoring NO levels and alleviating oxidative stress.

ß-Elemene is a major bioactive sesquiterpenoids compound isolated from the essential oils of Curcuma Wenyujin, a Chinese medicinal herb that treats tumor in clinics. However anti-atherosclerotic effects of ß-elemene have not been fully investigated in vivo. The objective of this study is to further elucidate the anti-atherosclerotic activities of ß-elemene in ApoE-/- mice. Staining techniques and immunohistochemistry were used to validate atherosclerosis. Serum lipids, plasma nitrite and nitrate were analyzed by colorometric methods. ROS and antioxidative enzymes were measured through kits. Proteome profiler array was performed to analyze atherosclerosis-related inflammatory Cytokine. Western blot was used for measuring various proteins expressions. These results revealed that ß-elemene inhibited atherosclerotic lesion size and increased stability of plaques in ApoE-/- mice by alleviating levels of vascular oxidative stress and preventing pro-inflammatory cytokine production. In addition ß-elemene maintained endothelial function by significantly improving plasma nitrite and nitrate levels and expression of phosphorylation-eNOS in vivo. ß-elemene also increased the production of the nitric oxide (NO) in human umbilical vein endothelial cells (HUVECs) and promoted phosphorylation of eNOSser1177 and Akt in vitro. In Conclusive, data revealed that ß-elemene attenuated atherosclerosis and enhanced stability of plaques at least partially through its antioxidative and anti-inflammatory features and protected against endothelial dysfunction in ApoE-/- mice.

Ovarian response prediction in controlled ovarian stimulation for IVF using anti-Müllerian hormone in Chinese women: A retrospective cohort study.

The predictive value of anti-Müllerian hormone (AMH) in Chinese women undergoing in vitro fertilization (IVF) treatment is data deficient. To determine the attributes of AMH in IVF, oocyte yield, cycle cancellation, and pregnancy outcomes were analyzed. All patients initiating their first IVF cycle with gonadotropin-releasing hormone agonist treatment in our center from October 2013 through December 2014 were included, except patients diagnosed with polycystic ovarian syndrome. Serum samples collected prior to IVF treatment were used to determine serum AMH levels. A total of 4017 continuous cycles were analyzed. The AMH level was positively correlated with the number of oocytes retrieved. Overall, AMH was significantly correlated with risk of cycle cancellation, poor ovarian response (POR, 3, or fewer oocytes retrieved) and high response (>15 oocytes), with an area under the curve (AUC) of 0.83, 0.89, and 0.82 respectively. An AMH cutoff of 0.6 ng/mL had a sensitivity of 54.0% and a specificity of 90.0% for the prediction of cycle cancellation, and cutoff of 0.8 ng/mL with a sensitivity of 55.0% and a specificity of 94.0% for the prediction of POR. Compared with AMH >2.0 ng/mL, patients with AMH < 0.6 ng/mL had a 53.6-fold increased risk of cancellation (P < 0.001), and AMH <0.80 ng/mL were 17.5 times more likely to experience POR (P < 0.001). However, AMH was less predictive of pregnancy and live birth, with AUCs of 0.55 and 0.53, respectively. Clinical pregnancy rate, ongoing pregnancy rate, and live birth rate per retrieval according to the AMH level (≤0.40, 0.41-0.60, 0.61-0.80, 0.81-1.00, 1.01-1.50, 1.51-2.00, and >2.00 ng/mL) showed no significant differences. Even with AMH≤0.4 ng/mL, 50.0% of all the patients achieved pregnancy and 34.8% of patients achieved live birth after transfer. Our results suggested that AMH is a fairly robust metric for the prediction of cycle cancellation and oocyte yield for Chinese women, but it is a relatively poor test for prediction of pregnancy outcomes. Patients with low levels of AMH still can achieve reasonable treatment outcomes and low AMH levels in isolation do not represent an appropriate marker for withholding fertility treatment.