Variation of Ferroptosis-Related Markers in HaCaT Cell Photoaging Models Induced by UVB.

Objective: To investigate the variation of ferroptosis-related markers in HaCaT cell photoaging models induced by ultraviolet-B (UVB). Methods: UVB-treated HaCaT cells served as the model (UVB group) for cellular photoaging, whereas untreated HaCaT cells served as the control group. HaCaT cells were exposed to UVB and the ferroptosis inhibitor Ferrostatin-1 (Fer-1) as part of the UVB+Fer-1 group, and co-cultured with the ferroptosis inducer Erastin as part of the UVB+Erastin group. Reactive oxygen species (ROS) detection kit and senescence-related ß galactosidase (SA-ß-gal) staining were used to evaluate the senescence of HaCaT cells. Lipid reactive oxygen species were detected by C11 BODIPY581/591 probe and mitochondrial morphology was observed by transmission electron microscopy. The mRNA expressions of glutathione peroxidase 4 (GPX4) and ferroptosis-suppressor-protein 1 (FSP1) were detected by real-time reverse transcription-PCR (RT-RCP), and the level of GPX4 protein was measured by immunofluorescence assay. Results: The UVB group had considerably greater levels of ROS, SA-ß-gal, and lipid reactive oxygen species than the control group. The UVB group's mitochondrial volume was reduced, the membrane density increased, and the mitochondrial crest decreased or even disappeared. GPX4 and FSP1 expression levels were similarly found to be lower in the UVB group. Furthermore, the positive rate of SA-ß-gal and lipid reactive oxygen species in the UVB+Fer-1 group was much lower than in the UVB group, but it was reverse in the UVB+Erastin group. This study showed that induced ferroptosis can aggravate aging, and vice versa. Conclusion: According to the findings, ferroptosis may be linked to UVB-induced skin photoaging, which could be attenuated by inhibition of ferroptosis.

Formulation and evaluation of a two-stage targeted liposome coated with hyaluronic acid for improving lung cancer chemotherapy and overcoming multidrug resistance.

Multidrug resistance (MDR) has emerged as a prominent challenge contributing to the ineffectiveness of chemotherapy in treating non-small cell lung cancer (NSCLC) patients. Currently, mitochondria of cancer cells are identified as a promising target for overcoming MDR due to their crucial role in intrinsic apoptosis pathway and energy supply centers. Here, a two-stage targeted liposome (HA/TT LP/PTX) was successfully developed via a two-step process: PTX-loaded cationic liposome (TT LP/PTX) were formulated by lipid film hydration & ultrasound technique, followed by further coating with natural anionic polysaccharide hyaluronic acid (HA). TT, an amphipathic polymer conjugate of triphenylphosphine (TPP)-tocopheryl polyethylene glycol succinate (TPGS), was used to modify the liposomes for mitochondrial targeting. The average particle size, zeta potential and encapsulation efficiency (EE%) of HA/TT LP/PTX were found to be 153 nm, -30.3 mV and 92.1% based on the optimal prescription of HA/TT LP/PTX. Compared to cationic liposome, HA-coated liposomes showed improved stability and safety, including biological stability in serum, cytocompatibility, and lower hemolysis percentage. In drug-resistant A549/T cells, HA was shown to improve the cellular uptake of PTX through CD44 receptor-mediated endocytosis and subsequent degradation by hyaluronidase (HAase) in endosomes. Following this, the exposure of TT polymer facilitated the accumulation of PTX within the mitochondria. As a result, the function of mitochondria in A549/T cells was disturbed, leading to an increased ROS level, decreased ATP level, dissipated MMP, and increased G2/M phase arrest. This resulted in a higher apoptotic rate and stronger anticancer efficacy.

Mechanism of Ba Zhen Tang Delaying Skin Photoaging Based on Network Pharmacology and Molecular Docking.

Purpose: To study the efficacy of Ba Zhen Tang in delaying skin photoaging and its potential mechanism based on network pharmacology and molecular docking. Methods: First, we screened the active components and targets of Ba Zhen Tang by Traditional Chinese Medicine Database and Analysis Platform (TCMSP) and The Universal Protein Resource (UniProt). The target genes of skin photoaging were obtained from GeneCards and GeneMap database. Then, we analyzed the protein-protein interaction (PPI) by STRING database. The network map was constructed by Cytoscape. Finally, we performed Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis by Metascape database. The molecular docking via Autodock Vina and Pymol. Furthermore, skin photoaging cellular models were established, and the effects of Ba Zhen Tang on ameliorating skin photoaging were investigated. Results: A total of 160 active ingredients in Ba Zhen Tang and 60 targets of Ba Zhen Tang for delaying skin photoaging were identified. By GO enrichment analysis, 1153 biological process entries, 45 cellular component entries and 89 molecular functional entries were obtained. A total of 155 signal pathways were obtained by KEGG analysis. Ba Zhen Tang is related to MAPK signaling pathway, TNF signaling pathway and AGE-RAGE signaling pathway in diabetic complications, etc., which directly affect the key nodes of photoaging. The molecular docking results showed that there was a certain affinity between the main compounds (kaempferol, quercetin, ß-sitosterol, naringenin) and core target genes (PTGS2, CASP3, MAPK1, MAPK3, TP53). Ba Zhen Tang-treated mouse serum inhibited the senescence and p16INK4a expression of human immortalized keratinocyte (HaCaT) cells irradiated by ultraviolet-B (UVB). Conclusion: Our study elucidated the potential pharmacological mechanism of Ba Zhen Tang in the treatment of photoaging through multiple targets and pathways. The therapeutic effects of Ba Zhen Tang on skin photoaging were validated in cellular models.

A network pharmacology-based study of the potential targets and mechanisms of action of Qibao Meiran Dan in delaying skin aging.

OBJECTIVE: The aim of this study was to use network pharmacology to explore the potential targets and mechanisms of action of Qibao Meiran Dan in relation to delaying skin aging. METHODS: The traditional Chinese medicine systems pharmacology database and analysis platform, and the traditional Chinese medicine integrated database, were used to screen the active ingredients and targets of Qibao Meiran Dan. The human gene database GeneCards and the gene database of the National Center for Biotechnology Information were jointly adopted to obtain skin aging-related target genes. The search tool for the retrieval of interacting genes/proteins (STRING) database was used for core analysis of protein-protein interaction. RESULTS: In total, 72 effective active ingredients, 273 action targets, 234 skin aging target genes, and 64 intersecting core targets were identified. GO enrichment analysis provided 393 biological process entries, and the KEGG analysis was represented by the tumor necrosis factor (TNF) signaling pathway, where the core targets of TNF-α and matrix metalloproteinase-1 (MMP-1) were enriched. The experimental results showed that cell morphology was clearer and more refractive in the Qibao Meiran Dan group than in the model group. CONCLUSION: Qibao Meiran Dan may regulate oxidative stress injury and collagen metabolism by downregulating the expression of TNF-α and MMP-1, thus slowing skin aging.

Artesunate treatment ameliorates ultraviolet irradiation-driven skin photoaging via increasing ß-catenin expression.

OBJECTIVE: Artesunate, a semi-synthetic derivative of artemisinin, exerts various pharmacological activities. Nevertheless, the effects of Art on skin photoaging remain unclear. Herein, we investigated whether Art ameliorated ultraviolet-irradiated skin photoaging in HaCaT cells and mice. METHODS: To construct skin photoaging cellular models, HaCaT cells were irradiated by UV (UVB, 20mJ/cm2) for 5 days. HaCaT cells were pretreated with three concentrations of Art (1, 5 and 20 µg/ml) for 2 h each day. After 5 days, cell senescence, ROS production, SOD levels, p16INK4a and ß-catenin expression, proliferation and apoptosis were detected in HaCaT cells. Effects of Art on normal cells were investigated. After sh-ß-catenin transfection or XAV-939 treatment, HaCaT cells were pretreated with 20 µg/ml Art and irradiated by UVB. After 5 days, skin photoaging was then observed. Furthermore, skin photoaging mouse models were established and the effects of Art and ß-catenin silencing on skin photoaging were investigated. RESULTS: Art treatment suppressed cell senescence, intracellular ROS production, p16INK4a expression and apoptosis and promoted proliferation and SOD and ß-catenin expression in UVB irradiated HaCaT cells. But Art had no toxic effects on normal cells. Silencing ß-catenin by sh-ß-catenin or XAV-939 exacerbated UVB irradiation-mediated cell senescence, apoptosis, and ROS production in HaCaT cells, which was ameliorated by Art treatment. The therapeutic effects of Art on skin photoaging were also confirmed in mouse models. CONCLUSIONS: These findings suggested that Art treatment alleviated UVB irradiation-driven skin photoaging through enhancing ß-catenin expression, which offered novel clues for pharmacological activity of Art.

The effect of Yang Yan Qing E Wan on senescent phenotypes and the expression of ß-catenin and p16INK4a in human skin fibroblasts.

This aim of this study was to observe the effect of Yang Yan Qing E Wan (YYQEW) on senescent phenotypes and the expression of ß-catenin and p16INK4a in the hydrogen peroxide (H2O2)-induced premature senescence of normal human skin fibroblasts (NHSFs). Primary normal human skin fibroblasts were randomly divided into a normal group, a blank group, a model group, and a YYQEW group. The cells of the model group and the YYQEW group were exposed to 150 µmol/L H2O2 for 2 h. The morphological changes of the cells were analyzed by microscopy and by kits used to estimate the activities of the senescence-associated ß-galactosidase (SA-ß-gal), reactive oxygen species (ROS), and superoxide dismutase (SOD). The outcomes revealed that dyeing rate proportion of SA-ß-gal was 2.78% ± 0.22% in the normal group, 2.83% ± 0.29% in the blank group, 37.58% ± 2.56% in the model group, and 28.39% ± 0.93% in the YYQEW group. The number of SA-ß-gal positive cells was thus significantly higher in the model group than in the normal or blank group. There were also fewer SA-ß-gal positive cells in the YYQEW group compared with the model group. The expression of ROS and p16INK4a in the model group increased significantly compared with that in the normal or blank groups, while the expression of ROS and p16INK4a in the YYQEW group decreased significantly compared with that in the model group. The expression of SOD and ß-catenin in the model group decreased significantly compared with that in the normal or blank group, and the expression of SOD and ß-catenin in the YYQEW group increased significantly compared with that in the model group. Overall, it was found that YYQEW was able to delay the senescence of NHSFs induced by H2O2 treatment by alleviating oxidative stress and regulating a number of senescence-related molecules, such as ß-catenin and p16INK4a.

First record of Leptospira and Blastocystis infections in captive flying squirrels (Trogopterus xanthipes) from Enshi County, China.

In traditional Chinese medicine, the feces of flying squirrels have long been used to promote blood circulation and relieve bodily stasis. However, the excrement of flying squirrels may harbor zoonotic agents that could be hazardous to public health. To understand the occurrence of bacterial and parasitic infections in this species, we investigated selected zoonotic pathogens including Leptospira and Blastocystis in the urine and feces of flying squirrels in China. Urine and fecal samples from flying squirrels were collected from a family-owned flying squirrel farm located in Enshi County, Hubei Province in China. Leptospira and Blastocystis DNA was extracted from the urine and feces of flying squirrels, and used as targets for PCR amplification, using different specific primers. PCR amplification and DNA sequencing showed that 4.4% (3/69) of flying squirrels were positive for Leptospira, while 30.4% (21/69) of the animals were positive for Blastocystis. Notably, 1.4% (1/69) of flying squirrels were found to be co-infected with Leptospira and Blastocystis. Sequence analyses allowed for the detection of 3 Blastocystis subtypes (ST1, ST3 and ST13), and mixed infections of Blastocystis subtype 1 and subtype 3 were found in 4.4% (3/69) of flying squirrels. Phylogenetic analysis of the 16S ribosomal RNA gene (rrs2), the flagellin B gene (flaB), and outer membrane lipoprotein lipL32 gene (LipL32) sequences indicated that the Leptospira species detected in the study was L. interrogans. We concluded that flying squirrels from central China were infected with Leptospira and Blastocystis, suggesting that these animals can be a source of infection for their owners, and using fresh excrement from this animal as traditional medicine could be risky to human health. To the best of our knowledge, this is the first report of Leptospira and Blastocystis infection in flying squirrels from Enshi County, China. Our findings provide new data on the epidemiology of these pathogens in this region.

Pseudomonas hutmensis sp. nov., a New Fluorescent Member of Pseudomonas putida Group.

A rod-shaped, Gram-negative and aerobic bacterium, strain XWS2, was isolated from rhizosphere soil of a camphor tree in Hubei University of Chinese Medicine Huangjiahu Campus, Wuhan, China. Cells grew at 4-37 °C (optimum 28 °C), pH 5.0-9.0 (optimum 7.0) and with 0-5% NaCl (optimum 1%). Colonies growing on tryptone soybean agar are round, beige in color and approximately 2 mm in diameter after 24 h incubation at 28 °C. Pellicle formation during liquid culture and strong fluorescent pigment production on King's B medium are typical features of strain XWS2. The genome of XWS2 is 6,170,117 bp, containing 5682 predicted genes and 4770 genes are functionally annotated. Phylogenetic analysis based on 16S rRNA sequence showed that strain XWS2 formed an independent branch within the Pseudomonas putida group, with P. putida NBRC 14164T (99.86% similarity) and P. alkylphenolica KL28T (99.36% similarity) as the most closely related type strains. Genome sequence analysis based on average nucleotide identity and digital DNA-DNA hybridization are below the threshold values for species delineation. Phenotypic characteristics, physiological and biochemical tests also supported the strain represents a separate novel species within the Pseudomonas genus. The name Pseudomonas hutmensis sp. nov. is proposed, with type strain XWS2T (= CCTCC AB 2018189 T = KACC 19898T).

[Bushen Huoxue Fang promotes the apoptosis of epithelial cells in the prostatic ductal system of rats with benign prostatic hyperplasia].

OBJECTIVE: To investigate the effects of Bushen Huoxue Fang (BSHX) on the apoptosis of epithelial cells in the prostatic ductal system of rats with benign prostatic hyperplasia (BPH) and its possible action mechanism. METHODS: One hundred 3- month-old male Wistar rats were randomly divided into four groups of equal number (control, castrated, BPH model, and BSHX). BPH models were made by subcutaneous injection of testosterone following castration; the rats in the BSHX group were treated intragastrically with BSHX at 2.34 g/ml after modeling, while those in the other two groups with equal volume of saline, all for 37 days. On the 38th day, all the rats were sacrificed and their prostates harvested for detection of the distribution of TGF-beta1 and alpha-actin and the count of positive cells in the prostatic ductal system by immunohistochemical staining. The apoptosis rate of epithelial cells in the prostatic ductal system was determined by TUNEL assay. RESULTS: The expression of TGF-beta1 was significantly increased in the rats of the BSHX group as compared with the BPH models in both the proximal prostatic duct ([15.28 +/- 4.30]% vs [36.42 +/- 8.10]%, P < 0.01) and the distal prostatic duct ([4.42 +/- 2.07]% vs [8.71 +/- 2.28 ]%, P < 0.05), while the expression of alpha-actin in the proximal duct was remarkably higher in the BSHX-treated rats than in the models ([28.14 +/- 7.43]% vs [18.28 +/- 4.07]%, P < 0.01), but lower than in the control animals ([33.57 +/- 6.85]%, P < 0.05). Compared with the control group, the BPH models and BSHX-treated rats both exhibited markedly decreased apoptosis of epithelial cells in the proximal prostatic duct ([39.42 +/- 9.20]% vs [3.86 +/- 1.34]%, P < 0.01, and [31.14 +/- 5.64]%, P < 0.01) and distal prostatic duct ([17.60 +/- 4.86]% vs [3.07 +/- 1.14]%, P < 0.01, and [12.37 +/- 2.25]%, P < 0.05). The apoptosis rate of epithelial cells in the prostatic ductal system was significantly higher in the BSHX-treated rats than in the BPH models (P < 0.01). CONCLUSION: By upregulating the expression of TGF-beta, BSHX can suppress the reduction of smooth muscle cells in the proximal prostatic duct, promote the apoptosis of prostatic epithelial cells, and thus effectively inhibit benign prostatic hyperplasia.

Study on traditional Chinese medicine theory of lung being connected with large intestine.

OBJECTIVE: The theory of lung being connected with large intestine, which is a major topic in Traditional Chinese Medicine (TCM), has guided clinical practice for thousands of years in China. METHODS: In this study, we analyzed the history, main contents, clinical application, and material basis of the theory, to attempt to improve the potential clinical significance of "lung being connected with large intestine" in China. RESULTS: The lung being connected with large intestine was first described in "Huang Di Nei Jing", and formed one of the basic theories of TCM. For thousands of years, the majority of TCM practitioners explored this theory continuously, leading to its development and use as an important theory in the guidance of TCM clinics In the last decade, researchers in the field of integrated TCM and Western medicine have studied clinical applications and biomedical mechanisms with experimental methods to explore the implications of the theory. CONCLUSION: With the further development of science and technology, research concerning the theory of lung being connected with large intestine will be greatly stimulated and contribute to the modernization of TCM.

The applications and obstacles of metabonomics in traditional chinese medicine.

In the recent years, a wide range of metabonomic technologies are widely used in the modern research of traditional chinese medicine (TCM). At present, the most prevailing methods for TCM research are mainly nuclear magnetic resonance (NMR), gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS). With these techniques, metabonomics will help to understand syndromes, efficacy and toxicity of TCM. However, every analytical technique has its advantages and drawbacks, and there exist some obstacles of its applications on TCM. So, we discuss metabonomics in TCM and analyze some problems of its applications to study TCM in recent years. We believe that with the further development of metabonomic analytical technology, especially multianalysed techniques, metabonomics will greatly promote TCM research and be beneficial to the modernization of TCM.

Anticancer, antioxidant and antimicrobial activities of the essential oil of Lycopus lucidus Turcz. var. hirtus Regel.

This study was designed to examine the anticancer, antioxidant and antimicrobial activities of the essential oil from Lycopus lucidus Turcz. var. hirtus Regel. The essential oil treatment to six human cancer cell lines resulted in a dose-dependent inhibition of cell growth. The cytotoxicity of the essential oil on liver carcinoma and breast cancer cell lines was significantly stronger than on other cell lines. The essential oil can induce apoptosis of the liver carcinoma cell line Bel-7402 and decrease the intracellular GSH level. The antioxidant effect of the essential oil was evaluated by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radical (OH) scavenging assays. The essential oil exhibited moderate antioxidant activity. The antimicrobial activity of the essential oil was evaluated against eight microorganisms using the disc diffusion and broth microdilution methods. The essential oil also showed moderate antimicrobial activity. These suggest that the essential oil could hold a good potential for use in the pharmaceutical industry.

[Effects of Guben Hutan Tongmai Recipe on expressions of macrophages and cell adhesion molecules of aortic endothelia in rats with syndrome of phlegm blocking blood vessel].

OBJECTIVE: To observe the effects of Guben Huatan Tongmai Recipe (GBHTTMR), a compound Chinese herbal recipe, on expressions of macrophages and cell adhesion molecules (CAMs) of aortic endothelia in rats with syndrome of phlegm blocking blood vessel, and to explore the pathogenesis of the phlegm-pathogen. METHODS: Fifty normal male Wistar rats, 7-week in age, were randomly divided into five groups: normal control group, untreated group, high-dose GBHTTMR-treated group, low-dose GBHTTMR-treated group and simvastatin-treated group, with 10 rats in each group. Syndrome of phlegm blocking blood vessel was induced in rats of the latter 4 groups by feeding the rats with high lipid diet. Levels of blood lipid were compared among the 5 groups. The expressions of macrophages and CAMs in aortic endothelia were tested by immunohistochemical staining method. RESULTS: The level of blood lipid, and the expressions of macrophages and CAMs showed statistical differences between the normal control group and the untreated group (P<0.01), and between the untreated group and the low-, high-dose GBHTTMR-treated and simvastatin-treated groups as well (P<0.05). CONCLUSIONS: GBHTTMR can decrease the level of serum cholesterol and triglycerides, and increase the level of high density lipoprotein. It also can inhibit the expressions of macrophages, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, P-selectin, and E-selectin.

[Expression of NF-kappaB and COX-2 mRNA in rats with phlegm obstruction due to lung-deficiency].

OBJECTIVE: To study the role of nuclear factor-kappa B (NF-kappaB) and cycloxygenase-2 (COX-2) in the onset of phlegm obstruction due to lung-deficiency in rats and the therapeutic mechanism of Huatan Recipe. METHODS: Twenty-four SD rats were randomly divided into normal group, model group and treatment group, with 8 rats in each group. The rats in the model group and treatment group were exposed to sulfur dioxide and cold wind to establish the rat model of phlegm obstruction due to lung-deficiency, and the rats in the treatment group were also treated with Huatan Recipe, a compound traditional Chinese medicine. The expression of NF-kappaB in the bronchial epithelial cells of the rats was tested with the method of immunohistochemistry, and the COX-2 mRNA in the lung tissues of the rats was measured by using reverse transcription-polymerase chain reaction. RESULTS: The expressions of NF-kappaB and COX-2 mRNA in rats of the model group were higher than those of the normal group (P<0.01), and the expressions of NF-kappaB and COX-2 mRNA in rats of the treatment group were obviously lower than those of the model group (P<0.01). CONCLUSION: The NF-kappaB and COX-2 play an important role in the onset of phlegm obstruction in rats. Huatan Recipe may prevent the development of phlegm obstruction by down-regulating the expressions of NF-kappaB and COX-2 mRNA.