Active ingredient and mechanistic analysis of traditional Chinese medicine formulas for the prevention and treatment of COVID-19: Insights from bioinformatics and in vitro experiments.

Coronavirus disease 2019 (COVID-19) is an acute infectious disease caused by a novel coronavirus. Traditional Chinese medicine (TCM) has been proven to have a potential curative effect on COVID-19. This study preliminarily analyzed the existing TCM prescription's key components and action mechanisms for preventing and treating COVID-19 using bioinformatic and experimental methods. Association and clustering analysis reveals that the "HQ + FF + BZ" drug combination had a strong correlation and confidence in 93 TCM prescriptions and may affect the progression of COVID-19 through inflammatory pathways such as the TNF signaling pathway. Further molecular docking revealed that quercetin has a higher affinity for IL6 and IL10 in the TNF signaling pathway associated with COVID-19. In vitro experiments demonstrated that quercetin could effectively reduce the levels of the inflammatory factor IL-6 and increase the anti-inflammatory factor IL-10, alleviating inflammation impact on cells. Our results provide a new understanding of the molecular mechanism of TCM prevention and treatment of COVID-19, which is helpful to the development of new diagnosis and treatment schemes for COVID-19.

Multichannel Ca2+ Generator for Synergistic Tumor Therapy via Intracellular Ca2+ Overload and Chemotherapy.

Ca2+ overload has attracted an increasing attention due to its benefit of precise cancer therapy, but its efficacy is limited by the strong Ca2+ excretion of cancer cells. Moreover, monotherapy of Ca2+ overload usually fails to treat tumors satisfactorily. Herein, we develop a multifunctional nanosystem that could induce Ca2+ overload by multipathway and simultaneously produce chemotherapy for synergistic tumor therapy. The nanosystem (CaMSN@CUR) is prepared by synthesizing a Ca-doped mesoporous silica nanoparticle (CaMSN) followed by loading the anticancer drug curcumin (CUR). CaMSN serves as the basis Ca2+ generator to induce Ca2+ overload directly in the intracellular environment by acid-triggered Ca2+ release, while CUR could not only exhibit chemotherapy but also facilitate Ca2+ release from the endoplasmic reticulum to the cytoplasm and inhibit Ca2+ efflux out of cells to further enhance Ca2+ overload. The in vitro and in vivo results show that CaMSN@CUR could exhibit a remarkable cytotoxicity against 4T1 cells and significantly inhibit tumor growth in 4T1 tumor-bearing mice via the synergy of Ca2+ overload and CUR-mediated chemotherapy. It is expected that the designed CaMSN@CUR has a great potential for effective tumor therapy.

Fermentation products of Danshen relieved dextran sulfate sodium-induced experimental ulcerative colitis in mice.

With the increased incidence and recognition, ulcerative colitis (UC) has become a global public health problem in the world. Although many immunosuppressant and biological drugs have been used for UC treatment, the cure rate is still very low. It is necessary to find some safe and long-term used medicine for UC cure. Recently, the Chinese traditional herb Danshen has been investigated in the treatment of UC. However, it is a limitation of Danshen that many of the active components in Danshen are not easily absorbed by the human body. Probiotics could convert macromolecules into smaller molecules to facilitate absorption. Thus, Lactobacillus rhamnosus (F-B4-1) and Bacillus subtillis Natto (F-A7-1) were screened to ferment Danshen in this study. The fermented Danshen products were gavaged in the dextran sulfate sodium (DSS)-induced UC model mice. Danshen had better results to attenuate symptoms of DSS-induced UC after fermented with F-B4-1 and F-A7-1. Loss of body weight and disease activity index (DAI) were reduced. The abnormally short colon lengths and colonic damage were recovered. And fermented Danshen had the better inhibitory effect than Danshen itself on pro-inflammatory cytokine expression during DSS-induced UC. The results indicated that compared with Danshen, fermented Danshen relieved DSS-induced UC in mice more effectively. Danshen fermented by probiotics might be an effective treatment to UC in clinic stage in the future.

The effect of folic acid deficiency on Mest/Peg1 in neural tube defects.

OBJECTIVE: Neural tube defects (NTDs) are one of the most common and serious birth defects in human beings caused by genetic and environmental factors. Folate insufficiency is involved in the occurrence of NTDs and folic acid supplementation can prevent NTDs occurrence, however, the underlying mechanism remains poorly understood. METHODS: We established cell and animal models of folic acid deficiency to detect the methylation modification and expression levels of genes by MassARRAY and real-time PCR, respectively. Results and conclusion: In the present study, we found firstly that in human folic acid-insufficient NTDs, the methylation level of imprinted gene Mest/Peg1 was decreased. By using a folic acid-deficient cell model, we demonstrated that Mest/Peg1 methylation was descended. Meanwhile, the mRNA level of Mest/Peg1 was up-regulated via hypomethylation modification under low folic acid conditions. Consistent with the results in cell models, Mest/Peg1 expression was elevated through hypomethylation regulation in folate-deficient animal models. Furthermore, the up-regulation of Mest/Peg1 inhibited the expression of Lrp6 gene, a crucial component of Wnt pathway. Similar results with Lrp6 down-regulation of fetal brain were verified in animal models under folic acid-deficient condition. Taken together, our findings indicated folic acid increased the expression of Mest/Peg1 via hypomethylation modification, and then inhibited Lrp6 expression, which may ultimately impact on the development of nervous system through the inactivation of Wnt pathway.

Improving microbial bioremediation efficiency of intensive aquacultural wastewater based on bacterial pollutant metabolism kinetics analysis.

How to effectively bioremediate aquacultural wastewater using microbes is an urgent issue for the application of aquaculture beneficial microorganisms. Purple non-sulfur bacteria (PNSB) are beneficial in preventing related pollution in aquaculture applications. An autochthonous PNSB Rhodobacter sphaeroides was employed in this study to explore an effective bioremediation strategy of aquacultural wastewater. The test bacterium showed high performance in the removal of ammonium (97.50% ± 0.78% of 42 mg L-1 NH4+-N) and phosphate (93.24% ± 0.71% of 50 mg L-1 PO43--P) in the synthetic wastewater, which are the two crucial indicators of the aquacultural wastewater bioremediation. The study also unveiled that the imbalanced ratio of nutrients in water was the principal reason for limiting the efficient bioremediation of shrimp-culture wastewater. Therefore, an effective microbial bioremediation strategy was proposed by comprehensively considering bacterial pollutant metabolism kinetics constants such as specific consumption yields of chemical oxygen demand (COD)/phosphorous and nitrogen/phosphorous. Finally, COD, total nitrogen (TN), total phosphorus (TP), and ammonium (NH4+-N) in the wastewater were examined, and the results showed that they all decreased to the acceptable values. In conclusion, this study suggested a novel method for improved bioremediation efficiency of aquacultural wastewater, and the findings revealed that this strategy is promising due to its characteristics to be used in various aquaculture wastewater types.

Calreticulin acts as an adjuvant to promote dendritic cell maturation and enhances antigen-specific cytotoxic T lymphocyte responses against non-small cell lung cancer cells.

Dendritic cell (DC)-based immunotherapy has promising for treatment of non-small cell lung cancer (NSCLC). Melanoma-associated antigen 3 (MAGE-A3) is a tumor-specific antigen and expressed in approximately 35-40% of NSCLC tissues. Calreticulin (CALR) is a protein chaperone and can enhance DC maturation and antigen presentation. In this study, we evaluated the adjuvant activity of CALR in human DC maturation and their capacity to induce MAGE-A3-specific CD8+ cytotoxic T lymphocyte (CTL) responses to NSCLC in vitro. Infection with recombinant Ad-CALR and/or Ad-MAGE-A3, but not with control Ads, induced CALR and/or MAGE-A3 expression in DCs. Infection with Ad-CALR significantly increased the percentages of CD80+, CD83+, CD86+ and HLA-DR+ DCs and IL-12 secretion, but reduced IL-10 production in DCs. Co-culture of autologous lymphocytes with DC-Ad-CALR or DC-Ad-CM significantly increased the numbers of induced CD8+ CTLs. The percentages of IFNγ-secreting CTLs responding to SK-LU-1 and NCI-H522 NSCLC, but not to non-tumor NL-20 cells in Ad-C-CTL, Ad-M-CTL and Ad-CM-CTL were significantly higher than that of DC-CTL and Ad-null-CTL. Ad-C-CTL, Ad-M-CTL and Ad-CM-CTL, but not control DC-CTL and Ad-null-CTL, induced higher frequency of MAGE-A3+HLA-A2+ NCI-H-522 cell apoptosis, but did not affect the survival of MAGE-A3+HLA-A2- SK-LU-1 and non-tumor NL20 cells in vitro. Treatment with anti-HLA-I antibody, but not with anti-HLA-II, dramatically diminished the cytotoxicity of Ad-CM-CTLs against NCI-H522 cells. Our data indicated that CALR acted as an adjuvant to promote DC maturation, which induced CTL development and enhanced MAGE-A3-specific CTL cytotoxicity against NSCLC.

Paclitaxel-hyaluronic nanoconjugates prolong overall survival in a preclinical brain metastases of breast cancer model.

Brain (central nervous system; CNS) metastases pose a life-threatening problem for women with advanced metastatic breast cancer. It has recently been shown that the vasculature within preclinical brain metastasis model markedly restricts paclitaxel delivery in approximately 90% of CNS lesions. Therefore to improve efficacy, we have developed an ultra-small hyaluronic acid (HA) paclitaxel nanoconjugate (∼5 kDa) that can passively diffuse across the leaky blood-tumor barrier and then be taken up into cancer cells (MDA-MB-231Br) via CD44 receptor-mediated endocytocis. Using CD44 receptor-mediated endocytosis as an uptake mechanism, HA-paclitaxel was able to bypass p-glycoprotein-mediated efflux on the surface of the cancer cells. In vitro cytoxicity of the conjugate and free paclitaxel were similar in that they (i) both caused cell-cycle arrest in the G2-M phase, (ii) showed similar degrees of apoptosis induction (cleaved caspase), and (iii) had similar IC50 values when compared with paclitaxel in MTT assay. A preclinical model of brain metastases of breast cancer using intracardiac injections of Luc-2 transfected MDA-MB-231Br cells was used to evaluate in vivo efficacy of the nanoconjugate. The animals administered with HA-paclitaxel nanoconjugate had significantly longer overall survival compared with the control and the paclitaxel-treated group (P < 0.05). This study suggests that the small molecular weight HA-paclitaxel nanoconjugates can improve standard chemotherapeutic drug efficacy in a preclinical model of brain metastases of breast cancer.

Anticancer activities of (-)-epigallocatechin-3-gallate encapsulated nanoliposomes in MCF7 breast cancer cells.

The chemopreventive actions exerted by green tea are thought to be due to its major polyphenol, (-)-epigallocatechin-3-gallate (EGCG). However, the low level of stability and bioavailability in the body makes administering EGCG at chemopreventive doses unrealistic. We synthesized EGCG encapsulated chitosan-coated nanoliposomes (CSLIPO-EGCG), and observed their antiproliferative and proapoptotic effect in MCF7 breast cancer cells. CSLIPO-EGCG significantly enhanced EGCG stability, improved sustained release, increased intracellular EGCG content in MCF7 cells, induced apoptosis of MCF7 cells, and inhibited MCF7 cell proliferation compared to native EGCG and void CSLIPO. The CSLIPO-EGCG retained its antiproliferative and proapoptotic effectiveness at 10 µM or lower, at which native EGCG does not have any beneficial effects. This study portends a potential breakthrough in the prevention or even treatment of breast cancer by using biocompatible and biodegradable CSLIPO-EGCG with enhanced chemopreventive efficacy and minimized immunogenicity and side-effects.

Gypenosides induce apoptosis by ca2+ overload mediated by endoplasmic-reticulum and store-operated ca2+ channels in human hepatoma cells.

Gypenosides (Gyps) are triterpenoid saponins contained in an extract from Gynostemma pentaphyllum Makino and reported to induce apoptosis in human hepatoma cells through Ca(2+)-implicated endoplasmic reticulum (ER) stress and mitochondria-dependent pathways. The mechanism underlying the Gyp-increased intracellular Ca(2+) concentration ([Ca(2+)]i) is unclear. Here, we examined Gyp-induced necrosis and apoptosis in human hepatoma HepG2 cells. Gyp-induced apoptotic cell death was accompanied by a sustained increase in [Ca(2+)]i level. Gyp-increased [Ca(2+)]i level was partly inhibited by removal of extracellular Ca(2+) by Ca(2+) chelator EGTA, store-operated Ca(2+) channel (SOC) inhibitor 2- aminoethoxydiphenyl borate (2-APB), and ER Ca(2+)-release-antagonist 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8). The strongest inhibitory effect was observed with TMB-8. EGTA, 2-APB, and TMB-8 also protected against Gyp-induced apoptosis in HepG2 cells. The combination of 2-APB and TMB-8 almost completely abolished the Gyp-induced Ca(2+) response and apoptosis. In contrast, the sarco/endoplasmic-reticulum-Ca(2+)-ATPase (SERCA) inhibitor thapsigargin slightly elevated Gyp-induced [Ca(2+)]i increase and apoptosis in HepG2 cells. Exposure to 300 µg/mL Gyp for 24 hours upregulated protein levels of inositol 1,4,5-trisphosphate receptor and SOC and downregulated that of SERCA for at least 72 hours. Thus, Gyp-induced increase in [Ca(2+)]i level and consequent apoptosis in HepG2 cells may be mainly due to enhanced Ca(2+) release from ER stores and increased store-operated Ca(2+) entry.

Brain histological changes in young mice submitted to diets with different ratios of n-6/n-3 polyunsaturated fatty acids during maternal pregnancy and lactation.

N-3 polyunsaturated fatty acids (n-3 PUFAs) are essential for brain development and function, but the appropriate quantity of dietary n-3 PUFAs and ratio of n-6/n-3 PUFAs have not been clearly determined. In this study, we investigated the effects of different dietary ratios of n-6/n-3 PUFAs on the brain structural development in mice and the expression of associated transcription factors. C57 BL/6J mice were fed with one of two categories of n-3 PUFA-containing diets (a flaxseed oil diet and a flaxseed/fish oil mixed diet) or an n-3 PUFA-deficient diet. For each of the n-3 PUFA diets, flaxseed oil or flaxseed/fish oil was combined with other oils to yield three different n-6/n-3 ratios, which ranged from 15.7:1 to 1.6:1. The feeding regimens began two months before mouse conception and continued throughout lactation for new pups. As compared with the n-3 PUFA-deficient diet, both the flaxseed oil n-3 PUFA diets and the flaxseed/fish oil n-3 PUFA diets significantly increased the expression levels of brain neuron-specific enolase, glial fibrillary acidic protein and myelin basic protein, somewhat dose-dependently, in new pup mice at 21 d and 42 d of age. The expression of PPAR-γ in the brains of pup mice was increased only at 7 d of age with the n-3 PUFA diet, and no changes in the expression of PPAR-α and PPAR-ß were found among all the diet groups. These results suggest that the higher intake amount of n-3 PUFAs with a low ratio of n-6/n-3 PUFAs at about 1-2:1, supplied during both maternal pregnancy and lactation, may be more beneficial for early brain development, and PPAR-γ may act in one of the pathways by which n-3 PUFAs promote early brain development.