Sevoflurane causes cognitive impairment by inducing iron deficiency and inhibiting the proliferation of neural precursor cells in infant mice.

AIMS: Numerous studies on animals have shown that exposure to general anesthetics in infant stage may cause neurocognitive impairment. However, the exact mechanism is not clear. The dysfunction of iron metabolism can cause neurodevelopmental disorders. Therefore, we investigated the effect of iron metabolism disorder induced by sevoflurane (Sev) on cognitive function and the proliferation of neural precursor cells (NPCs) and neural stem cells (NSCs) in infant mice. METHODS: C57BL/6 mice of postnatal day 14 and neural stem cells NE4C were treated with 2% Sev for 6 h. We used the Morris water maze (MWM) to test the cognitive function of infant mice. The proliferation of NPCs was measured using bromodeoxyuridine (BrdU) label and their markers Ki67 and Pax6 in infant brain tissues 12 h after anesthesia. Meanwhile, we used immunohistochemical stain, immunofluorescence assay, western blot, and flow cytometer to evaluate the myelinogenesis, iron levels, and cell proliferation in cortex and hippocampus or in NE4C cells. RESULTS: The results showed that Sev significantly caused cognitive deficiency in infant mice. Further, we found that Sev inhibited oligodendrocytes proliferation and myelinogenesis by decreasing MBP and CC-1 expression and iron levels. Meanwhile, Sev also induced the iron deficiency in neurons and NSCs by downregulating FtH and FtL expression and upregulating the TfR1 expression in the cortex and hippocampus, which dramatically suppressed the proliferation of NSCs and NPCs as indicated by decreasing the colocalization of Pax6+ and BrdU+ cells, and caused the decrease in the number of neurons. Interestingly, iron supplementation before anesthesia significantly improved iron deficiency in cortex and hippocampus and cognitive deficiency induced by Sev in infant mice. Iron therapy inhibited the decrease of MBP expression, iron levels in neurons and oligodendrocytes, and DNA synthesis of Pax6+ cells in hippocampus induced by Sev. Meanwhile, the number of neurons was partially recovered in hippocampus. CONCLUSION: The results from the present study demonstrated that Sev-induced iron deficiency might be a new mechanism of cognitive impairment caused by inhaled anesthetics in infant mice. Iron supplementation before anesthesia is an effective strategy to prevent cognitive impairment caused by Sev in infants.

Facile process ofHibiscusmucilage polymer formulation usingHibiscus rosa-sinensisleaves to treat second-degree burn and excision wounds.

Mucilage is a sticky substance found in various plants and microorganisms and is made up of proteins and polysaccharides. Mucilage fromHibiscus rosa sinensisisis a complex polysaccharide traditionally used to treat different skin diseases. In our study, we fabricated mucilage polymer fromHibiscus rosa sinensisleaves and evaluated its potential application in second-degree burns and excision wounds. The physical properties of Hibiscus mucilage (HM) polymer were demonstrated by using Ultraviolet-visible absorption spectroscopy, x-ray diffraction, Fourier transform infrared spectroscopy, dynamic light scattering, Scanning electron microscopy, Brunauer-Emmett-Tellerand, Swelling ratio. The human cell lines WI-38, and HaCaT have been used forin-vitroexperiments like MTT, scratch wound, BrdU, ROS scavenging assays, and western blot analysis. The results of the MTT, scratch-wound, and BrdU assay indicated that the HM polymer is nontoxic in nature and also enhances both the properties of cellular migration and proliferation, respectively. On the other hand, the result of the ROS scavenging assay suggested that HM polymer enhances the antioxidant activity of cells while the western blot analysis designated that the HM polymer treatment caused downregulation of the pro-inflammatory cytokine IFN-γand upregulation of the pAkt (Serine 473) protein, and TGF-ß1 signaling pathway. Therefore, allin-vitroexperimental studies recommended that HM polymer is biocompatible and has antioxidant and anti-inflammatory effects. In thein vivoexperiment, second-degree burns and excision wounds were created on the dorsal surface of male BALB/c mice. After the sixth day of HM polymer treatment have developed new tissue, hair follicles, blood vessels,α-SMA, and Collagen type-1 fiber on the burn and excision wound area while the 11th day of HM polymer treatment cured the wound area significantly. Therefore, it could be contemplated that HM polymer is a potential agent for treating different wounds in the near future.

Constitutive cell proliferation and neurogenesis in the organum vasculosum lamina terminalis and subfornical organ of adult rats.

Neurogenesis continues throughout adulthood in the subventricular zone, hippocampal subgranular zone, and the hypothalamic median eminence (ME) and the adjacent medio-basal hypothalamus. The ME is one of the circumventricular organs (CVO), which are specialized brain areas characterized by an incomplete blood-brain barrier and, thus, are involved in mediating communication between the central nervous system and the periphery. Additional CVOs include the organum vasculosum laminae terminalis (OVLT) and the subfornical organs (SFO). Previous studies have demonstrated that the ME contains neural stem cells (NSCs) capable of generating new neurons and glia in the adult brain. However, it remains unclear whether the OVLT and SFO also contain proliferating cells, the identity of these cells, and their ability to differentiate into mature neurons. Here we show that glial and mural subtypes exhibit NSC characteristics, expressing the endogenous mitotic maker Ki67, and incorporating the exogenous mitotic marker BrdU in the OVLT and SFO of adult rats. Glial cells constitutively proliferating in the SFO comprise NG2 glia, while in the OVLT, both NG2 glia and tanycytes appear to constitute the NSC pool. Furthermore, pericytes, which are mural cells associated with capillaries, also contribute to the pool of cells constitutively proliferating in the OVLT and SFO of adult rats. In addition to these glial and mural cells, a fraction of NSCs containing proliferation markers Ki67 and BrdU also expresses the early postmitotic neuronal marker doublecortin, suggesting that these CVOs comprise newborn neurons. Notably, these neurons can differentiate and express the mature neuronal marker NeuN. These findings establish the sensory CVOs OVLT and SFO as additional neurogenic niches, where the generation of new neurons and glia persists in the adult brain.

Periplaneta Americana (L.) extract activates the ERK/CREB/BDNF pathway to promote post-stroke neuroregeneration and recovery of neurological functions in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Periplaneta americana (L.) (PA) has been used in traditional Chinese medicine for thousands of years for the effect of invigorating blood circulation and removing blood stasis. Modern pharmacological research shown that PA extract exhibits promising effects in promoting wound healing and regeneration, as well as in brain diseases such as Parkinson's disease (PD). However, whether it is effective for neuroregeneration and neurological function recovery after stroke still unknown. AIM OF THE STUDY: This study aims to investigate the potential effect of PA extract to promote brain remodeling through the activation of endogenous neurogenesis and angiogenesis, in addition, preliminary exploration of its regulatory mechanism. METHODS: Firstly, BrdU proliferation assay and immunofluorescence (IF) staining were used to evaluate the effect of PA extract on the neurogenesis and angiogenesis in vitro and in vivo. Subsequently, the effects of PA extract on brain injury in stroke rats were assessed by TTC and HE. While mNSS score, adhesive removal test, rota-rod test, and morris water maze test were used to assess the impact of PA extract on neurological function in post-stroke rats. Finally, the molecular mechanisms of PA extract regulation were explored by RNA-Seq and western blotting. RESULTS: The number of BrdU+ cells in C17.2 cells, NSCs and BMECs dramatically increased, as well as the expression of astrocyte marker protein GFAP and neuronal marker protein Tuj-1 in C17.2 and NSCs. Moreover, PA extract also increased the number of BrdU+DCX+, BrdU+GFAP+, BrdU+CD31+ cells in the SGZ area of transient middle cerebral artery occlusion model (tMCAO) rats. TTC and HE staining revealed that PA extract significantly reduced the infarction volume and ameliorated the pathological damage. Behavioral tests demonstrated that treatment with PA extract reduced the mNSS score and the time required to remove adhesive tape, while increasing the time spent on the rotarod. Additionally, in the morris water maze test, the frequency of crossing platform and the time spent in the platform quadrant increased. Finally, RNA-Seq and Western blot revealed that PA extract increased the expression of p-ERK, p-CREB and BDNF. Importantly, PA extract mediated proliferation and differentiation of C17.2 and NSCs reversed by the ERK inhibitor SCH772984 and the BDNF inhibitor ANA-12, respectively. CONCLUSION: Our study demonstrated that PA extract promoted neurogenesis and angiogenesis by activating the CREB/ERK signaling pathway and upregulating BDNF expression, thereby recovering neurological dysfunction in post-stroke.

Electroacupuncture promotes neurogenesis in the dentate gyrus and improves pattern separation in an early Alzheimer's disease mouse model.

BACKGROUND: Impaired pattern separation occurs in the early stage of Alzheimer's disease (AD), and hippocampal dentate gyrus (DG) neurogenesis participates in pattern separation. Here, we investigated whether spatial memory discrimination impairment can be improved by promoting the hippocampal DG granule cell neogenesis-mediated pattern separation in the early stage of AD by electroacupuncture (EA). METHODS: Five familial AD mutations (5 × FAD) mice received EA treatment at Baihui and Shenting points for 4 weeks. During EA, mice were intraperitoneally injected with BrdU (50 mg/kg) twice a day. rAAV containing Wnt5a shRNA was injected into the bilateral DG region, and the viral efficiency was evaluated by detecting Wnt5a mRNA levels. Cognitive behavior tests were conducted to assess the impact of EA treatment on cognitive function. The hippocampal DG area Aß deposition level was detected by immunohistochemistry after the intervention; The number of BrdU+/CaR+ cells and the gene expression level of calretinin (CaR) and prospero homeobox 1(Prox1) in the DG area of the hippocampus was detected to assess neurogenesis by immunofluorescence and western blotting after the intervention; The gene expression levels of FZD2, Wnt5a, DVL2, p-DVL2, CaMKII, and p-CaMKII in the Wnt signaling pathway were detected by Western blotting after the intervention. RESULTS: Cognitive behavioral tests showed that 5 × FAD mice had impaired pattern separation (P < 0.001), which could be improved by EA (P < 0.01). Immunofluorescence and Western blot showed that the expression of Wnt5a in the hippocampus was decreased (P < 0.001), and the neurogenesis in the DG was impaired (P < 0.001) in 5 × FAD mice. EA could increase the expression level of Wnt5a (P < 0.05) and promote the neurogenesis of immature granule cells (P < 0.05) and the development of neuronal dendritic spines (P < 0.05). Interference of Wnt5a expression aggravated the damage of neurogenesis (P < 0.05), weakened the memory discrimination ability (P < 0.05), and inhibited the beneficial effect of EA (P < 0.05) in AD mice. The expression level of Wnt pathway related proteins such as FZD2, DVL2, p-DVL2, CAMKII, p-CAMKII increased after EA, but the effect of EA was inhibited after Wnt5a was knocked down. In addition, EA could reduce the deposition of Aß plaques in the DG without any impact on Wnt5a. CONCLUSION: EA can promote hippocampal DG immature granule cell neogenesis-mediated pattern separation to improve spatial memory discrimination impairment by regulating Wnt5a in 5 × FAD mice.

[Effect of electroacupuncture on proliferation and differentiation of endogenous neural stem cells and Jagged1/Notch1 pathway in hippocampus of APP/PS1 model mice].

OBJECTIVE: To investigate the effect of electroacupuncture(EA) stimulation on proliferation and diffe-rentiation of endogenous neural stem cells as well as Jagged1/Notch1 pathway in AD model mice, so as to explore its mechanism underlying amelioration of AD. METHODS: A total of 40 6-week-old male APP/PS1 transgenic AD mice were randomly divided into EA group (n=20) and AD model group ( n=20), and other 20 normal C57BL/6J mice of the same age were used as the normal control group. The mice in the EA group received EA (10 Hz, 2 mA) at "Baihui"(GV20), "Fengfu"(GV16) and bilateral "Shenshu" (BL23) for 20 min, once daily, 6 days a week for 16 weeks. The mice's learning-memory ability was detected by Morris water maze tests. The Aß senile plaques in the hippocampal CA1 region were detected by Congo red staining, the immunofluorescence double label of BrdU, neuronal nuclear antigen (NeuN) and astrocyte specific protein GFAP in dentate gyrus of hippocampus was performed for detecting the proliferation and differentiation of the endogenous neural stem cells. The expression levels of Nestin (neuron specific protein) and GFAP were detected by Western blot, and those of Jagged1 and Notch1 mRNAs and proteins in the hippocampus were detected by real-time fluorescence quantifative PCR and Western blot. RESULTS: Compared with the normal control group, the escape latencies at 2nd, 3rd and 4th day, and Aß senile plaques were significantly increased (P<0.05, P<0.01), whereas the platform crossing times and time spent in the target quadrant, the expression levels of Jagged1 mRNA and Nestin protein were remarkably down-regulated (P<0.05) in the model group. Following EA intervention, the escape latencies at the 3rd and 4th day, Aß senile plaques, immunofluorescence density of BrdU/GFAP, and GFAP protein expression were pronouncedly decreased (P<0.05, P<0.01), while the platform crossing times, platform quadrant residence time, immunofluorescence density of BrdU/NeuN, expression levels of Jagged1 and Notch1 mRNAs and proteins and Nestin protein evidently increased (P<0.05, P<0.01), suggesting an enhancement of proliferation and diffe-rentiation of endogenous neural stem cells into neurons and a suppression of the proliferation and differentiation towards astrocytes in the hippocampus. CONCLUSION: EA at GV20, GV16 and BL23 can improve the learning-memory ability, promote the proliferation and differentiation of endogenous neural stem cells towards neurons and inhibit the proliferation and differentiation of endogenous neural stem cells towards astrocytes in the hippocampus, which may be achieved by regulating Jagged1/Notch1 pathway.

Identification and Quantification of Proliferating Cells in Skeletal Muscle of Glutamine Supplemented Low- and Normal-Birth-Weight Piglets.

One way to improve the growth of low-birth-weight (LBW) piglets can be stimulation of the cellular development of muscle by optimized amino acid supply. In the current study, it was investigated how glutamine (Gln) supplementation affects muscle tissue of LBW and normal-birth-weight (NBW) piglets. Longissimus and semitendinosus muscles of 96 male piglets, which were supplemented with 1 g Gln/kg body weight or alanine, were collected at slaughter on day 5 or 26 post natum (dpn), one hour after injection with Bromodeoxyuridine (BrdU, 12 mg/kg). Immunohistochemistry was applied to detect proliferating, BrdU-positive cells in muscle cross-sections. Serial stainings with cell type specific antibodies enabled detection and subsequent quantification of proliferating satellite cells and identification of further proliferating cell types, e.g., preadipocytes and immune cells. The results indicated that satellite cells and macrophages comprise the largest fractions of proliferating cells in skeletal muscle of piglets early after birth. The Gln supplementation somewhat stimulated satellite cells. We observed differences between the two muscles, but no influence of the piglets' birth weight was observed. Thus, Gln supplements may not be considered as effective treatment in piglets with low birth weight for improvement of muscle growth.

[Effect of Morus alba extract sanggenon C on growth and proliferation of glioblastoma cells].

Glioblastoma is the most common primary cranial malignancy, and chemotherapy remains an important tool for its treatment. Sanggenon C(San C), a class of natural flavonoids extracted from Morus plants, is a potential antitumor herbal monomer. In this study, the effect of San C on the growth and proliferation of glioblastoma cells was examined by methyl thiazolyl tetrazolium(MTT) assay and 5-bromodeoxyuridinc(BrdU) labeling assay. The effect of San C on the tumor cell cycle was examined by flow cytometry, and the effect of San C on clone formation and self-renewal ability of tumor cells was examined by soft agar assay. Western blot and bioinformatics analysis were used to investigate the mechanism of the antitumor activity of San C. In the presence of San C, the MTT assay showed that San C significantly inhibited the growth and proliferation of tumor cells in a dose and time-dependent manner. BrdU labeling assay showed that San C significantly attenuated the DNA replication activity in the nucleus of tumor cells. Flow cytometry confirmed that San C blocked the cell cycle of tumor cells in G_0/G_1 phase. The soft agar clone formation assay revealed that San C significantly attenuated the clone formation and self-renewal ability of tumor cells. The gene set enrichment analysis(GSEA) implied that San C inhibited the tumor cell division cycle by affecting the myelocytomatosis viral oncogene(MYC) signaling pathway. Western blot assay revealed that San C inhibited the expression of cyclin through the regulation of the MYC signaling pathway by lysine demethylase 4B(KDM4B), which ultimately inhibited the growth and proliferation of glioblastoma cells and self-renewal. In conclusion, San C exhibits the potential antitumor activity by targeting the KDM4B-MYC axis to inhibit glioblastoma cell growth, proliferation, and self-renewal.

Characteristics of the Cytotoxicity of Taraxacum mongolicum and Taraxacum formosanum in Human Breast Cancer Cells.

Breast cancer is a highly heterogeneous disease that has been clinically divided into three main subtypes: estrogen receptor (ER)- and progesterone receptor (PR)-positive, human epidermal growth factor receptor 2 (HER 2)-positive, and triple-negative breast cancer (TNBC). With its high metastatic potential and resistance to endocrine therapy, HER 2-targeted therapy, and chemotherapy, TNBC represents an enormous clinical challenge. The genus Taraxacum is used to treat breast cancer in traditional medicine. Here, we applied aqueous extracts from two Taraxacum species, T. mongolicum and T. formosanum, to compare their potential antitumor effects against three human breast cancer cell lines: MDA-MB-231 (ER-, PR-, and HER2-), ZR-75-1 (ER+, PR+/-, and HER2-), and MCF-7 (ER+, PR+, and HER2-). Our results show that T. mongolicum exerted cytotoxic effects against MDA-MB-231 cells, including the induction of apoptosis, the reduction of cell proliferation, the disruption of the mitochondrial membrane potential, and/or the downregulation of the oxygen consumption rate. Both T. mongolicum and T. formosanum decreased cell migration and colony formation in the three cell-lines and exerted suppressive effects on MCF-7 cell proliferation based on metabolic activity and BrdU incorporation, but an enhanced proliferation of ZR-75-1 cells based on BrdU incorporation. T. formosanum induced ribotoxic stress in MDA-MB-231and ZR-75-1 cells; T. mongolicum did not. In summary, these findings suggest that T. mongolicum showed greater cytotoxicity against all three tested breast cancer cell lines, especially the TNBC MDA-MB-231 cell line.

Christmas Tree Bio-Waste as a Power Source of Bioactive Materials with Anti-Proliferative Activities for Oral Care.

According to the American Cancer Society, roughly 54,000 new cases of oral cavity or oropharyngeal cancers have been detected in the United States of America in 2021, and they will cause about 10,850 deaths. The main therapies for cancer management, such as surgery and radio- and chemotherapy, have some own benefits, albeit they are often destructive for surrounding tissues; thus, deep investigations into non-surgical treatments for oral cavities are needed. Biologically active compounds (BACs) extracted from European Spruce needles were analyzed to determine the total phenolic and flavonoid content and were used as additional ingredients for oral hygiene products. An anti-proliferation investigation was carried out using extracts containing BACs with the use of several cell lines (cancer and a normal one). ESI-MS studies on BACs showed that luteolin, a natural flavonoid compound with anti-tumorigenic properties against various types of tumors, is the predominant component of the extracts. MTT, BrdU, and LIVE/DEAD studies demonstrated that BAC extracts obtained from Christmas tree needles possess anticancer properties against squamous cell carcinoma (with epithelial origins). We proved that BAC extracts contain high amounts of luteolin, which induces cytotoxicity toward cancer cells; along with their high selectivity, robustness, and nontoxicity, they are very promising materials in oral health applications.

Investigation of Naoluoxintong on the neural stem cells by facilitating proliferation and differentiation in vitro and on protecting neurons by up-regulating the expression of nestin in MCAO rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The classic traditional Chinese compound Naoluoxintong (NLXT) has been proven an effective remedy for ischemic stroke (IS). The protective effect of NLXT on neural stem cells (NSCs), however, remains unclear. AIM OF THE STUDY: To investigate the protective effect of NLXT on NSCs in rats with middle cerebral artery occlusion (MCAO) and the effect of Nestin expression in vivo. MATERIALS AND METHODS: Sprague-Dawley (SD) rats were randomly divided into three groups: the sham-operated group, the MCAO model group and the NLXT group. The MCAO model in rats was established by modified Longa wire embolization method. The sham-operated group, the model group and the NLXT groups were divided into three subgroups according to the sampling time points of 1 d, 3 d and 7 d after successful model-making. Immunofluorescence staining, including bromodeoxyuridine (BrdU)/glial fibrillary acidic protein (GFAP), ß-tubulinIII/GFAP, BrdU/doublecortin (DCX) and BrdU/neuronal nuclei (NeuN), was used to detect the proliferation and survival of NSCs in the hippocampal after drug administration. Protein expression of Nestin, DCX, GFAP and NeuN in the hippocampal was detected by Western blot (WB). RESULTS: Immunofluorescence experiment of Nestin labeled: on the first day, a few Nestin-positive cells were found in the hippocampal DG area. Afterwards, the number of Nestin-labeled positive cells in the model group increased, while the number of cells in the sham group did not fluctuate significantly. The number of positive cells in each administration group increased more than that in the model and normal group. ß-tubulin III/GFAP double-labeled: a small amount of double labeled cells was expressed in the normal group, and the number subsequently fluctuated little. In the model group, ß-tubulin III/GFAP positive cells increased initially after acute ischemia, and gradually decreased afterwards. In the NLXT-treated group, ß-Tubulin III positive cells were significantly increased on day 1, 3 and 7, while GFAP positive cells had little change. BrdU/DCX double-labeled: initially, a small number of BrdU/DCX-labeled positive cells were observed in the normal group and the model group, but there was no increasing trend over time. The positive cells in the NLXT group increased over time, and those in the seven-day group were significantly higher than those in the one-day and three-day groups. BrdU/NEUN double-labeled: in the normal group, BrdU/NEUN positive cells were enriched and distributed regularly. The number of positive cells in the model group was small and decreased gradually with time, and the decrease was most obvious on the third day. The number of positive cells in the NLXT group was significantly higher than that in the model group, and the number of positive cells in the seven-day group was significantly higher than that in the one-day and three-day groups. WB results reflected those three proteins, Nestin, NeuN and DCX, showed an increase in expression, except GFAP, which showed a decreasing trend. CONCLUSIONS: Preliminarily, NLXT can promote the migration and differentiation of NSCs. It may have a protective effect on the brain by promoting repair of brain tissue damage through upregulation of Nestin after IS.

Isoquinoline Alkaloids from Coptis chinensis Franch: Focus on Coptisine as a Potential Therapeutic Candidate against Gastric Cancer Cells.

Gastric cancer (GC) has high incidence rates and constitutes a common cause of cancer mortality. Despite advances in treatment, GC remains a challenge in cancer therapy which is why novel treatment strategies are needed. The interest in natural compounds has increased significantly in recent years because of their numerous biological activities, including anti-cancer action. The isolation of the bioactive compounds from Coptis chinensis Franch was carried out with the Centrifugal Partition Chromatography (CPC) technique, using a biphasic solvent system composed of chloroform (CHCl3)-methanol (MeOH)-water (H2O) (4:3:3, v/v) with an addition of hydrochloric acid and trietylamine. The identity of the isolated alkaloids was confirmed using a high resolution HPLC-MS chromatograph. The phytochemical constituents of Coptis chinensis such as berberine, jatrorrhizine, palmatine and coptisine significantly inhibited the viability and growth of gastric cancer cell lines ACC-201 and NCI-N87 in a dose-dependent manner, with coptisine showing the highest efficacy as revealed using MTT and BrdU assays, respectively. Flow cytometry analysis confirmed the coptisine-induced population of gastric cancer cells in sub-G1 phase and apoptosis. The combination of coptisine with cisplatin at the fixed-ratio of 1:1 exerted synergistic and additive interactions in ACC-201 and NCI-N87, respectively, as determined by means of isobolographic analysis. In in vivo assay, coptisine was safe for developing zebrafish at the dose equivalent to the highest dose active in vitro, but higher doses (greater than 10 times) caused morphological abnormalities in larvae. Our findings provide a theoretical foundation to further studies on more detailed mechanisms of the bioactive compounds from Coptis chinensis Franch anti-cancer action that inhibit GC cell survival in in vitro settings.

Curcumin induces thermotolerance by reducing oxidative stress, apoptosis, and inflammation in buffalo mammary epithelial cells under heat shock conditions.

The epithelial cell is the main basic unit of the udder in which milk synthesis takes place. Curcumin is well known for its antioxidant, anti-apoptotic, and anti- inflammatory properties. The present study was performed to test whether in vitro curcumin supplementation can alleviate the unfavorable impact of hyperthermia on buffalo mammary epithelial cells (BuMECs). The spontaneously immortalized BuMECs were divided into 7 groups (n = 9); 1) unstressed BuMECs (negative control, 37 °C); 2) BuMECs exposed to hyperthermia without curcumin treatment (positive control); 3-7) BuMECs cultured with different concentrations of curcumin (5, 10, 20, 40 and 60 µM), respectively, followed by hyperthermic exposure (42ºC) for 1 h and then returned to 37ºC. Changes in viability (MTT assay), proliferation (BrdU colorimetric immunoassay) and concentrations of antioxidant enzymes, CAT, and SOD (ELISA) of BuMECs were recorded. The gene expression study was performed using qRT-PCR. Lower concentrations of curcumin (5, 10 µM) maintained viability, enhanced proliferation, and content of antioxidant enzymes of heat stressed BuMECs. Curcumin induced thermotolerance and antioxidant status by upregulating the expression of antioxidants genes, anti-apoptotic genes and heat shock proteins in heat stressed BuMECs compared to the positive control group. Besides, curcumin reduced apoptosis and inflammation in BuMECs exposed to hyperthermia by downregulating the expression of genes and transcriptional factors associated with apoptosis and inflammatory immune response. The results reveal the potential roles of curcumin in eliminating the negative impact of hyperthermia on BuMECs by regulating the pathways of apoptosis, inflammation, and oxidative stress.

Persistent popliteal lymphatic muscle cell coverage defects despite amelioration of arthritis and recovery of popliteal lymphatic vessel function in TNF-Tg mice following anti-TNF therapy.

While rheumatoid arthritis patients and tumor necrosis factor transgenic (TNF-Tg) mice with inflammatory-erosive arthritis display lymphatic drainage deficits, the mechanisms responsible remain unknown. As ultrastructural studies of joint-draining popliteal lymphatic vessels (PLVs) in TNF-Tg mice revealed evidence of lymphatic muscle cell (LMC) damage, we aimed to evaluate PLV-LMC coverage in TNF-Tg mice. We tested the hypothesis that alpha smooth muscle actin (αSMA)+ PLV-LMC coverage decreases with severe inflammatory-erosive arthritis, and is recovered by anti-TNF therapy facilitated by increased PLV-LMC turnover during amelioration of joint disease. TNF-Tg mice with established disease received anti-TNF monoclonal antibody (mAb) or placebo IgG isotype control mAb therapy (n = 5) for 6-weeks, while wild-type (WT) littermates (n = 8) received vehicle (PBS). Bromodeoxyuridine (BrdU) was also administered daily during the treatment period to monitor PLV-LMC turnover. Effective anti-TNF therapy was confirmed by longitudinal assessment of popliteal lymph node (PLN) volume via ultrasound, PLV contraction frequency via near-infrared imaging of indocyanine green, and ankle bone volumes via micro-computed tomography (micro-CT). Terminal knee micro-CT, and ankle and knee histology were also performed. PLVs were immunostained for αSMA and BrdU to evaluate PLV-LMC coverage and turnover, respectively, via whole-mount fluorescent microscopy. Anti-TNF therapy reduced PLN volume, increased talus and patella bone volumes, and reduced tarsal and knee synovial areas compared to placebo treated TNF-Tg mice (p < 0.05), as expected. Anti-TNF therapy also increased PLV contraction frequency at 3-weeks (from 0.81 ± 1.0 to 3.2 ± 2.0 contractions per minute, p < 0.05). However, both anti-TNF and placebo treated TNF-Tg mice exhibited significantly reduced αSMA+ PLV-LMC coverage compared to WT (p < 0.05). There was no correlation of αSMA+ PLV-LMC coverage restoration with amelioration of inflammatory-erosive arthritis. Similarly, there was no difference in PLV-LMC turnover measured by BrdU labeling between WT, TNF-Tg placebo, and TNF-Tg anti-TNF groups with an average of < 1% BrdU+ PLV-LMCs incorporated per week. Taken together these results demonstrate that PLV-LMC turnover in adult mice is limited, and that recovery of PLV function during amelioration of inflammatory-erosive arthritis occurs without restoration of αSMA+ LMC coverage. Future studies are warranted to investigate the direct and indirect effects of chronic TNF exposure, and the role of proximal inflammatory cells on PLV contractility.

Transition milk stimulates intestinal development of neonatal Holstein calves.

Colostrum stimulates gastrointestinal development. Similar to colostrum, transition milk (TM; the first few milkings after colostrum) contains elevated nutrient levels and bioactive components not found in milk replacer (MR), albeit at lower levels than the first colostrum. We hypothesized that feeding neonatal calves TM, compared with MR, for 4 d following colostrum at birth would further stimulate intestinal development. Holstein bull calves were fed 2.8 L of colostrum within 20 min of birth, allocated to 1 of 11 blocks based on birth date and body weight (BW), randomly assigned to MR (n = 12) or TM (n = 11) treatments within block, and fed treatments 3 times per day. Milk from milkings 2, 3, and 4 (TM) of cows milked 2 times daily was pooled by milking number and fed at 1.89 L per feeding; milking 2 was fed at feedings 2 through 5, milking 3 at feedings 6 through 8, and milking 4 at feedings 9 through 12. TM was not pasteurized and contained 17% solids, 5% fat, 7% protein, 4% lactose, and 20 g of IgG per liter on average, whereas MR (as fed) contained 15% solids, 4% protein, 3% fat, 6% carbohydrate, and no IgG. Refusals were similar, so calves fed TM consumed 1.0 Mcal of metabolizable energy per day more than those fed MR. On the morning of d 5, calves were injected i.v. with 5 mg of bromodeoxyuridine per kg of BW and slaughtered 130 min later; then, intestinal sections were excised. Feeding TM, instead of MR, doubled villus length, villus width, villus to crypt ratio, and mucosal length in all intestinal sections, increased submucosal thickness 70% in the proximal and mid jejunum, and tended to increase submucosal thickness in duodenum and ileum. Mucosal surface area was also increased in both the ileum and mid jejunum when feeding TM by 19 and 36%, respectively. Treatment did not alter crypt depth. Bromodeoxyuridine labeling was increased 50% by TM compared with MR in the cells along the epithelium of the crypts and within the villi of all sections, indicating that TM increased cell proliferation compared with MR. Calves fed TM gained more BW than calves fed MR and had improved cough, fecal, nose, and ear scores. We conclude that feeding TM for 4 d following an initial feeding of colostrum stimulates villus, mucosal, and submucosal development in all sections of the small intestine in the first few days of life and improves health and growth.

Anticancer Effects of Vitis vinifera L. Mediated Biosynthesized Silver Nanoparticles and Cotreatment with 5 Fluorouracil on HT-29 Cell Line.

The aim of this study was to evaluate the anticancer effects of biosynthesized silver nanoparticles (Vv-AgNPs) from grape (Vitis vinifera L.) seed aqueous extract, alone or in combination with 5-Fluorouracil (5-FU) on HT-29 cell line. Vv-AgNPs were characterized by techniques such as UV-vis spectrophotometer (surface plasmon peak 454 nm), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX). HT-29 cells were treated with different concentrations (0-80 µg/mL for MTT) and (0-20 µg/mL for BrdU) of Vv-AgNPs alone and combined with (200 µg/mL) 5-FU for 72 h. The cytotoxic effects were analyzed by [3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl tetrazolium bromide] (MTT) assay (IC50 values 13.74 and 5.35 µg/mL, respectively). Antiproliferative effects were examined 5-bromo-2'-deoxyuridine (BrdU) assay (IC50 values 9.65 and 5.00 µg/mL, respectively). Activation of caspase-3 and protein expression levels of p53 were determined by Western blotting analysis. It was observed that Vv-AgNPs significantly increased the cleavage of the proapoptotic proteins caspase 3 and obviously enhanced the expression of p53 in a dose-dependent manner. The increased amount of total oxidant status (TOS) in the 10 µg/mL Vv-AgNPs + 5-FU treatment group, despite the increasing amount of total antioxidant status (TAS), caused an increase in Oxidative Stress Index (OSI) compared to the control. In this study, it has been shown in vitro that the use of successfully biosynthesized Vv-AgNPs in combination with 5-FU exhibits synergistic cytotoxic, antiproliferative, apoptotic, and oxidative effects against HT-29 cell line.

Hesperidin Reduces Memory Impairment Associated with Adult Rat Hippocampal Neurogenesis Triggered by Valproic Acid.

Treatment with valproic acid (VPA) deteriorates hippocampal neurogenesis, which leads to memory impairment. Hesperidin (Hsd) is a plant-based bioflavonoid that can augment learning and memory. This study aimed to understand the effect of Hsd on the impairment of hippocampal neurogenesis and memory caused by VPA. The VPA (300 mg/kg) was administered by intraperitoneal injection twice daily for 14 days, and Hsd (100 mg/kg/day) was administered by oral gavage once a day for 21 days. All rats underwent memory evaluation using the novel object location (NOL) and novel object recognition (NOR) tests. Immunofluorescent staining of Ki-67, BrdU/NeuN, and doublecortin (DCX) was applied to determine hippocampal neurogenesis in cell proliferation, neuronal survival, and population of the immature neurons, respectively. VPA-treated rats showed memory impairments in both memory tests. These impairments resulted from VPA-induced decreases in the number of Ki-67-, BrdU/NeuN-, and DCX-positive cells in the hippocampus, leading to memory loss. Nevertheless, the behavioral expression in the co-administration group was improved. After receiving co-administration with VPA and Hsd, the numbers of Ki-67-, BrdU/NeuN-, and DCX-positive cells were improved to the normal levels. These findings suggest that Hsd can reduce the VPA-induced hippocampal neurogenesis down-regulation that results in memory impairments.

Glutamine supplementation stimulates cell proliferation in skeletal muscle and cultivated myogenic cells of low birth weight piglets.

Muscle growth of low birth weight (LBW) piglets may be improved with adapted nutrition. This study elucidated effects of glutamine (Gln) supplementation on the cellular muscle development of LBW and normal birth weight (NBW) piglets. Male piglets (n = 144) were either supplemented with 1 g Gln/kg body weight or an isonitrogeneous amount of alanine (Ala) between postnatal day 1 and 12 (dpn). Twelve piglets per group were slaughtered at 5, 12 and 26 dpn, one hour after injection with Bromodeoxyuridine (BrdU, 12 mg/kg). Muscle samples were collected and myogenic cells were isolated and cultivated. Expression of muscle growth related genes was quantified with qPCR. Proliferating, BrdU-positive cells in muscle sections were detected with immunohistochemistry indicating different cell types and decreasing proliferation with age. More proliferation was observed in muscle tissue of LBW-GLN than LBW-ALA piglets at 5 dpn, but there was no clear effect of supplementation on related gene expression. Cell culture experiments indicated that Gln could promote cell proliferation in a dose dependent manner, but expression of myogenesis regulatory genes was not altered. Overall, Gln supplementation stimulated cell proliferation in muscle tissue and in vitro in myogenic cell culture, whereas muscle growth regulatory genes were barely altered.

Potential Effect of Pseudevernia furfuracea (L.) Zopf Extract and Metabolite Physodic Acid on Tumour Microenvironment Modulation in MCF-10A Cells.

Lichens comprise a number of unique secondary metabolites with remarkable biological activities and have become an interesting research topic for cancer therapy. However, only a few of these metabolites have been assessed for their effectiveness against various in vitro models. Therefore, the aim of the present study was to assess the effect of extract Pseudevernia furfuracea (L.) Zopf (PSE) and its metabolite physodic acid (Phy) on tumour microenvironment (TME) modulation, focusing on epithelial-mesenchymal transition (EMT), cancer-associated fibroblasts (CAFs) transformation and angiogenesis. Here, we demonstrate, by using flow cytometry, Western blot and immunofluorescence microscopy, that tested compounds inhibited the EMT process in MCF-10A breast cells through decreasing the level of different mesenchymal markers in a time- and dose-dependent manner. By the same mechanisms, PSE and Phy suppressed the function of Transforming growth factor beta (TGF-ß)-stimulated fibroblasts. Moreover, PSE and Phy resulted in a decreasing level of the TGF-ß canonical pathway Smad2/3, which is essential for tumour growth. Furthermore, PSE and Phy inhibited angiogenesis ex ovo in a quail embryo chorioallantoic model, which indicates their potential anti-angiogenic activity. These results also provided the first evidence of the modulation of TME by these substances.

YYFZBJS ameliorates colorectal cancer progression in ApcMin/+ mice by remodeling gut microbiota and inhibiting regulatory T-cell generation.

BACKGROUND: Progression of Colorectal cancer (CRC) is influenced by single or compounded environmental factors. Accumulating evidence shows that microbiota can influence the outcome of cancer immunotherapy. T cell, one of the main populations of effector immune cells in antitumor immunity, has been considered as a double-edged sword during the progression of CRC. Our previous studies indicate that traditional Chinese herbs (TCM) have potential anticancer effects in improving quality of life and therapeutic effect. However, little is known about the mechanism of TCM formula in cancer prevention. METHODS: Here, we used C57BL/6 J ApcMin/+ mice, an animal model of human intestinal tumorigenesis, to investigate the gut bacterial diversity and their mechanisms of action in gastrointestinal adenomas, and to evaluate the effects of Yi-Yi-Fu-Zi-Bai-Jiang-San (YYFZBJS) on of colon carcinogenesis in vivo and in vitro. Through human-into-mice fecal microbiota transplantation (FMT) experiments from YYFZBJS volunteers or control donors, we were able to differentially modulate the tumor microbiome and affect tumor growth as well as tumor immune infiltration. RESULTS: We report herein, YYFZBJS treatment blocked tumor initiation and progression in ApcMin/+ mice with less change of body weight and increased immune function. Moreover, diversity analysis of fecal samples demonstrated that YYFZBJS regulated animal's natural gut flora, including Bacteroides fragilis, Lachnospiraceae and so on. Intestinal tumors from conventional and germ-free mice fed with stool from YYFZBJS volunteers had been decreased. Some inflammation' expression also have been regulated by the gut microbiota mediated immune cells. Intestinal lymphatic, and mesenteric lymph nodes (MLN), accumulated CD4+ CD25+ Foxp3 positive Treg cells were reduced by YYFZBJS treatment in ApcMin/+ mice. Although YYFZBJS had no inhibition on CRC cell proliferation by itself, the altered Tregs mediated by YYFZBJS repressed CRC cancer cell growth, along with reduction of the phosphorylation of ß-catenin. CONCLUSIONS: In conclusion, we demonstrated that gut microbiota and Treg were involved in CRC development and progression, and we propose YYFZBJS as a new potential drug option for the treatment of CRC. Video abstract.