The Human Host Defense Peptide LL-37 Overexpressed in Lung Cell Lines by Methanolic Extract of Valeriana officinalis

Abstract The present study investigated the effects of valerian methanolic extract and valerenic acid on the expression of LL-37 gene and protein in A549 and MRC5 line cells. After preparing Valerian seeds, sowing them in March 2020, and harvesting the rhizome in October 2020, the extract was prepared from the valerian rhizome by maceration method. Valerian acid content was determined using high performance liquid chromatography (HPLC). Two cell lines (A549 and MRC-5) were used to study the effects of valerian extract, and the MTT test was used to evaluate cell viability. The expression of LL-37 mRNA and protein was assessed by Real-Time PCR and western blot, respectively. In vivo safety assessments and histopathological analysis were also conducted. Data was analyzed by Graphpad Prism 8 software. Valerian methanolic extract and valerenic acid upregulated the LL-37 mRNA and protein expression in both treated cell lines. Valerenic acid showed a greater effect on upregulating LL-37 expression than valerian methanolic extract. A549 cells were more sensitive to valerian methanolic extract compared to MRC5 cells, and its cell viability was reduced. Furthermore, liver and kidney-related safety assessments showed that valerian methanolic extract had no toxic effects. In general, it was concluded that the methanolic extract of valerian as well as the resulting valerenic acid as the most important component of the extract has the ability to upregulate LL-37expression. Therefore, methanolic extract of valerian and valerenic acid can be considered for improving the immune system.

Dexmedetomidine Leads to the Mitigation of Myocardial Ischemia/Reperfusion-Induced Acute Lung Injury in Diabetic Rats Via Modulation of Hypoxia-Inducible Factor-1α

ABSTRACT Introduction: The objective of this study is to investigate the protective mechanism of dexmedetomidine (Dex) in myocardial ischemia/reperfusion (MIR)-induced acute lung injury (ALI) of diabetic rats by inhibiting hypoxia-inducible factor-1α (HIF-1α). Methods: Initially, healthy male Sprague Dawley rats were treated with streptozocin to induce diabetes. Then, three weeks after the induction, Dex or lentiviral vector (LV)-HIF-1α was injected into the rats 30 minutes prior to the MIR modeling. After four weeks, lung tissues were harvested for pathological changes observation and the wet/dry weight (W/D) ratio determination. Afterwards, oxidative stress indicators and pro-inflammatory factors were measured. In addition, HIF-1α expression was assessed by immunohistochemistry and western blot analysis. Results: Dex could suppress inflammatory cell infiltration, improve lung tissue structure, reduce pathological score and the W/D ratio, and block oxidative stress and inflammatory response in MIR-induced ALI of diabetic rats. Besides, Dex could also inhibit HIF-1α expression. Moreover, Dex + LV-HIF-1α reversed the protective role of Dex on diabetic MIR-induced ALI. Conclusion: Our study has made it clear that Dex inhibited the upregulation of HIF-1α in diabetic MIR-induced ALI, and thus protect lung functions by quenching the accumulation of oxygen radical and reducing lung inflammatory response.

Expression of lncRNA DLEU1 in non-small cell lung cancer and its effect on metastasis / 中国肿瘤临床

Objective: To investigate the expression pattern and clinical significance of long non-coding RNA deleted in lymphocytic leu-kemia 1 (DLEU1) in non-small cell lung cancer (NSCLC), and to further evaluate its effect on tumor metastasis. Methods: Paired cancer and adjacent tissues were obtained from 42 patients with NSCLC that underwent surgical resection of cancer in The Third Xiangya Hos-pital of Central South University from January 2008 to December 2012, at the Department of Cardiothoracic Surgery. The expression features of DLEU1 in NSCLC tissue were detected by real-time quantitative polymerase chain reaction assay. Statistical methods were used to analyze the relationship between the expression of DLEU1 and the clinicopathological features and the survival time of pa-tients with NSCLC. Effects of DLEU1 on migration and invasion of NSCLC cells were evaluated by in vitro wound healing and cell inva-sion assays, respectively. Western blot was performed to investigate the protein levels of E-cadherin, N-cadherin, and Vimentin, which are the biomarkers of epithelial to mesenchymal transition (EMT). Results: Of the 42 NSCLC samples, DLEU1 expression was up-regulat-ed in 35 samples (83.33%) and down-regulated in 7 samples (16.67%). The expression level of DLEU1 in NSCLC tissue was 2.11 times that in the adjacent tissues (P<0.05). The expression of DLEU1 in four NSCLC cell lines (A549, H1299, SPCA1, and H358) was higher than that in normal lung 16HBE epithelial cells, and of these, A549 cells had the highest DLEU1 expression (P<0.05). Highly expressed DLEU1 was positively correlated with lymph-node metastasis (P<0.05), but was not significantly associated with other parameters, in-cluding patient gender, age, smoking history, primary tumor size, histological grade, and the TNM stage. The survival time of patients with high DLEU1 expression was significantly shorter than that of patients with low DLEU1 expression (P<0.05). In vitro interference with DLEU1 expression significantly inhibited the migration and invasion of A549 and SPCA1 cells compared with that of the control group (P<0.05). Upon interference with DLEU1 expression , protein levels of E-cadherin increased, whereas those of N-cadherin and Vi-mentin decreased in A549 cells in comparison with the control group. Conclusions: The expression of DLEU1 is up-regulated in NSCLC tissues and cell lines, correlates with lymph-node metastasis and survival times in patients with NSCLC, and promotes tumor cell migra-tion and invasion by regulating EMT, suggesting that DLEU1 may be a potential therapeutic target for NSCLC.

Updated review of resistance to neuromuscular blocking agents

Since neuromuscular blocking agents (NMBAs) were introduced to the surgical field, they have become almost mandatory for the induction and maintenance of anesthesia. However, resistance to NMBAs can develop in certain pathological states, such as central nerve injury, burns, and critical illnesses. During such pathological processes, quantitative and qualitative changes occur in the physiology of acetylcholine and the acetylcholine receptor (AChR) at the neuromuscular junction. Up-regulation of AChR leads to changes in the pharmacokinetics and pharmacodynamics of NMBA. As NMBA resistance may result in problems during anesthesia, it is of utmost importance to understand the mechanisms of NMBA resistance and their associations with pathological status to maintain adequate neuromuscular relaxation. This review presents the current knowledge of pharmacokinetic and pharmacodynamic changes and pathological status associated with NMBA resistance.

Expression of syntaxin 8 in glioma tissue and its clinical significance / 实用医学杂志

Objective To investigate the expression of syntaxin 8(STX8)in glioma and its clinical signif-icance. Methods Specimens of glioma were collected from 57 patients at Beijing Renhe Hospital from May 2013 to December 2015. 57 pieces of glioma tissue were used as a study group ,12 of which were Ⅰ+ Ⅱ(low grade) and the rest 45 were Ⅲ+Ⅳ;normal brain tissues from 15 individuals were used as a control group. Real-time PCR,immunohistochemistry,and Western blot were used to detect expression of STX8. Results As compared with the normal brain tissue ,the mRNA expression of STX8 was significantly increased in glioma tissue ,with a relative expression volume of 1.6855 ± 0.07124 in low grade and 2.8207 ± 0.0692 in high grade tissues,there was significant differences between the two groups;and the difference was also significant as compared with the control group(P < 0.05). The results of immunohistochemistry showed that the expression of STX8 was higher in glioma tissue than in normal tissue. Western Blot showed that the expression of STX8 protein was significantly higher in glioma than in normal tissue(P<0.05);the relative expression volume of STX8 was 2.271 ± 0.1621 in low grade tissue and 4.937 ± 0.1851 in high grade tissue,with a significant difference between the two groups;the difference was also significant as compared with the control group(P<0.05). The correlation analysis showed that higher STX8 expression in glioma was not significantly related to gender,age and pathological types,but there was a significant difference between pathological stages. Conclusion STX8 has abnormal high expression in glioma,which may be closely related with the occurrence and development of glioma.

Upregulation of PP1 Expression in Hippocampus Impairs Long-term Spatial Memory in Rats.

Aims: Either down-regulation of protein kinases or up-regulation of protein phosphatases weakens the level of neuronal protein phosphorylation and affects the function of neuron. Low-function and/or low-content of protein phosphatases in the hippocampus neurons is proposed to be a possible causative factor to the impairment of spatial memory ability. We show here that upregulation of PP1(protein phosphatase1) expression in hippocampus correlates with the decline of the spatial-memory retention ability in rat brain. Study Design: In the present study, 30 adult Wistar rats were tested in a one trial stepdown test to assess normal and impaired memory retention, examining protein phosphatases expression in their hippocampi and analysing both relationship. Place and Duration of Study: Department of Pathophysiology, Medical College in Wuhan University of Science and Technology (WUST), between June 2010 and July 2011. Methodology: To examine which rats have lower ability of learning and memory, thirty Wistar rats, male, 5-7 months old (360~450g), were trained and tested in step-down inhibitory avoidance task. R129d, R126d, R123d, Cdk5 (8) & CREB antibodies were respectively used to detect the content of PP1, PP2B, PP2A, Cdk5 & CREB. Densitometric analysis was used to quantitate the blots.Equal variance T test was used to analyze the escape latency and error from the two group rats, and the immunoblotting data from two groups of rat hippocampus proteins. Results: 72h after the fourth trail, MMI (mild memory impairmemt) rats showed performance markedly worse than that of the NC (normal control) rats ( latency shorter, <150sec; error more, ≥1 vs latency, 300sec; error, 0times; Both P<<0.01). From the learning and retention curves of the both groups of rats in Fig. 1, author found that in the first step-down train, the error times in MMI rats were also more than those in NC rats (P< 0.05), indicating that the ability of learning new material in MMI rats is worse than that in NC rats. The results showed that the contents of PP1 and PP-2B in MMI rats have increased or decreased ~50% (P<0.01) or ~40% (P<0.01) respectively compared with NC rats; Whereas the expression of PP2A in both groups of rats showed no obvious difference, implying that PP1 and/or PP2B might participate in the regulation of learning and memory in the rat brain. In addition, we all found that the middle degree of adverse correlation between the expression of PP1 and the latency of MMI & NC rats (-0.618, P<0.001, two tails), and the low degree of positive correlation between the expression of PP-2B and the latency of MMI & NC rats (0.381, P<0.05, two tails), indicating that the increase of PP1 content in itself might impress in part the memory ability of rats. Conclusion: It is concluded that upregulation of PP1 content may mediate worsened memory retention in rat brain.

The influence of microRNA-184 transfection on the biological behavior of human umbilical vein endothelial cells in vitro / 中华实验眼科杂志

Background Previous studies showed that microRNA-184 (miR-184) might inhibit the biological function of vascular endothelial cells.In ophthalmology,to determine the inhibitory effect of miR-184 on angiogenesis is of clinical significance for the prevention and treatment of corneal inflammation,retinal and choroidal neovascularization.Objective This study was to verify the influence of miR-184 transfection on the proliferation,migration and angiogenic ability of HUVECs and explore its mechanism.Methods HUVECs line was cultured in vitro and divided into 4 groups.No transfection regent and RNA molecule were added in the medium of the blank control group;only transfection regent was added in the blank transfection group; hsa-miR-184 mimic/negative was added in the negative control group,and Cy3 labeled hsa-miR-184 mimic-lipofectamine 2 000 mixture was added in the miR-184 transfection group.The proliferation of HUVECs (absorbance,A490) was detected by MTT.The transmembrane cell number was counted by Transwell insert to evaluate the migration ability of HUVECs.A three dimensional cultural system of cells was constructed on the matrigel,and tube formation number of HUVECs was assessed.Results Positive HUVECs for Cy3 labeled siR-RiboTM + hposome showed red fluorescence,with the optimal transfecting concentration ＞ 100 nmol/L.The relative expression levels of miR-184 were 1 524.10±385.89 and 1.00±0.05 in the miR-184 transfection group and the negative control group,showing a significant difference (t=-7.894,P＜0.01).The A490value of HUVECs was 0.50±0.04 and the number of migrating cells through the transwell membrane was (55.40±5.86)/field in the miR-184 transfection group,and they are significantly reduced in comparison with (0.62±0.04) and (83.40±5.59)/field in the negative control group (t =5.639,7.730,all at P＜ 0.01).A significant difference was found in the total branch points of capillary tubes between the miR-184 transfection group and negative control group ([13.33 ± 2.08]/field versus [22.00 ± 2.00]/field) (t =5.511,P＜ 0.05).Conclusions MiR-184 can suppress the proliferation,migration and tubing of HUVECs after transfection in vitro.MiR-184 participates in the regulation of angiogenesis.

Expression of procaspase 3 and activated caspase 3 and its relevance in hormone-responsive gallbladder carcinoma chemotherapy

BACKGROUND/AIMS: The higher incidence of gallbladder cancer (GBC) in females has been accredited to the involvement of hormones. The clinical implications of sex hormone receptors in GBC are well established. Cysteine proteases (such as caspase-3-9, etc.) are known to play a central role in the apoptotic pathway. Of these, the downstream enzyme caspase-3 is often activated in the apoptotic pathway. The aim of this work was to examine the status of apoptosis (which directly correlated with the level of active caspase-3) in hormone-responsive GBC. METHODS: We used 10 androgen receptor (AR)-positive, 14 estrogen receptor (ER)-positive, 12 HER/neu-positive, eight triple positive, and 10 triple negative malignant GBC human tissue samples. We isolated the total cellular protein from tumor tissues and carried out Western blotting using antipro-caspase-3 and anti-activated caspase-3 antibodies. RESULTS: ER and HER/neu-positive GBC exhibited high caspase-3 activity and low procaspase-3 activity, whereas AR-positive GBC showed no significant level of apoptosis. We also evaluated the apoptosis status of triple positive GBC and triple negative GBC, and found significant apoptosis in triple positive GBC. CONCLUSIONS: The results indicate that ER and HER/neu-positive GBCs had active apoptosis, whereas AR-positive GBC was highly resistant to apoptosis.

The effect of overexpression HMGA2 on proliferation and cell cycle in human growth hormone-secreting pituitary tumor cells / 重庆医学

Objective To explore the effects of over-expression high-mobility-group A2(HMGA2) on proliferation and cell cycle in primary cultured human growth hormone-secreting pituitary tumor cells .Methods The protein expression of HMGA2 in growth hormone-secreting pituitary tumor cells transfected by HMGA2 overexpression plasmid (transfection group) were detected by Western blot .The effects of the HMGA2 overexpression on cell proliferation ,cell cycle were measured by (cell counting kit-8 , CCK-8) assay and flow cytometry respectively .Results HMGA2 protein of transfection group was higher than the blank load transfection group(tranfectied with pcDNA3 .1 plasmid) after 24 h (P<0 .05) .To compare with the blank load transfection group , cells overexpression of HMGA2 could drastically increase the ability of proliferation at 24 ,48 ,72 ,96 h and increase the S phase rati-o of cell cycle and decrease the G1 phase ratio of cell cycle ,the difference was statistically significant (P<0 .05) .Conclusion Over-expression of HMGA2 increases pituitary tumor cells proliferation ability and promote cells G 1 phase progress .

Upregulation of hsa-miR-125b in HTLV-1 asymptomatic carriers and HTLV-1-associated myelopathy/tropical spastic paraparesis patients

The retrovirus human T lymphotropic virus type 1 (HTLV-1) promotes spastic paraparesis, adult T cell leukaemia and other diseases. Recently, some human microRNAs (miRNAs) have been described as important factors in host-virus interactions. This study compared miRNA expression in control individuals, asymptomatic HTLV-1 carriers and HTLV-1 associated myelopathy (HAM)/tropical spastic paraparesis patients. The proviral load and Tax protein expression were measured in order to characterize the patients. hsa-miR-125b expression was significantly higher in patients than in controls (p = 0.0285) or in the HAM group (p = 0.0312). Therefore, our findings suggest that miR-125b expression can be used to elucidate the mechanisms of viral replication and pathogenic processes.

Changes in cytosolic Ca2+ levels correspond to fluctuations of lactate levels in crosstalk of astrocyte-neuron cell lines.

Neurons and astrocytes differentially express isoenzymes of lactate dehydrogenase (LDH). The metabolic consequences for the variations in mRNA expression of LDH isoenzyme subtypes in neurons and astrocytes control cerebral vasoregulation. Moreover, cellular signalling consequences for functional neurovascular control may also be dependent on LDH isoenzyme subtype profiles. Initial computer simulations revealed glutamate-induced calcium waves in connected astrocytes, and showed concomitant changes in the expression of nitric oxide synthase (NOS) and lactic acid metabolism. To validate these findings, the nature and extent of glutamate-dependent signalling crosstalk in murine cell lines were investigated through correlated lactate levels and calcium upregulation. Neuro2A and C8D1A cells were separately treated with timed supernatant extracts from each other and their LDH1 and LDH5 isoenzyme responses were recorded. Western blot analysis showed LDH1/LDH5 isoenzyme ratio in the astrocytes to be positively correlated with Neuro2A-derived lactate levels estimated by the amplitude of 1.33-ppm spectral peak in 1H-NMR, and LDH1/LDH5 isoenzyme ratio in neurons is negatively correlated with C8D1A-derived lactate levels. Significant modulations of the calcium-responsive protein pCamKII levels were also observed in both cell lines, particularly correlations between pCamKII and lactate in C8D1A cells, thus explaining the calcium dependence of the lactate response. Together, these observations indicate that lactate is a key indicator of the metabolic state of these cell types, and may be a determinant of release of vasoregulatory factors.

Adaptive response: some underlying mechanisms and open questions: [review]

Organisms are affected by different DNA damaging agents naturally present in the environment or released as a result of human activity. Many defense mechanisms have evolved in organisms to minimize genotoxic damage. One of them is induced radioresistance or adaptive response. The adaptive response could be considered as a nonspecific phenomenon in which exposure to minimal stress could result in increased resistance to higher levels of the same or to other types of stress some hours later. A better understanding of the molecular mechanism underlying the adaptive response may lead to an improvement of cancer treatment, risk assessment and risk management strategies, radiation protection, e.g. of astronauts during long-term space flights. In this mini-review we discuss some open questions and the probable underlying mechanisms involved in adaptive response: the transcription of many genes and the activation of numerous signaling pathways that trigger cell defenses - DNA repair systems, induction of proteins synthesis, enhanced detoxification of free radicals and antioxidant production.

Pharmacological characteristics of homologous upregulation of nicotinic ?_7 receptors in brain / 中国药理学通报

10 ?mol?L -1) increased the binding sites of [ 125I] ?-Bgt significantly (P0.05).Conclusion Chronic treatment of choline, nicotine or methyllycaconitine can upregulate the nicotinic ? 7 receptors at certain doses. The effect of choline on upregulation of ? 7 receptors is different from that of nicotine.

Delayed Up-regulation of Vascular Endothelial Growth Factor and flk-1 after Global Cerebral Ischemia in Mongolian Gerbil: Possible roles in neuroangiogenesis?

BACKGROUND: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and a potent mediator of vascular permeability. Flk-1, one of the receptors for VEGF, is important in vascular development. Increased expression of VEGF is related with reactive astrogliosis, which stimulates the proliferation of neural progenitor cells. VEGF expression increases in the acute phase of cerebral ischemia, however the expression of VEGF together with flk-1 in subacute stage is still unknown. This study is done to demonstrate the spatial/cellular patterns of expression for VEGF/flk-1 up to subacute stages and to find out the role of VEGF in ischemia. METHODS: Transient global ischemia was induced by a 10 min-occlusion/reperfusion of the bilateral carotid arteries in the Mongolian gerbil. Immunohistochemistry and western blot were performed to ensure the expression of VEGF and flk-1 on the day 1, 3, 7, 14, and 28. RESULTS: Both VEGF and flk-1 initially increased at day1, and decreased at day 3. Thereafter, VEGF gradually increased again to the initial level at day 7 and to the peak level after day 14. Flk-1 showed a peak expression at day 14, and then decreased at day 28. Immunohistochemical staining for VEGF showed immunoreactivity mainly on the cytoplasm of neurons and endothelium in cortex and hippocampus at day 1, and neuron, endothelium, and glial cell from day 14 to 28. The distribution and chronological patterns of flk-1 expression were similar to that of VEGF expression. CONCLUSIONS: We suggest that global cerebral ischemia can induce a delayed up-regulation of VEGF and flk-1, which may be associated with neuroangiogenesis and repair process.

Up-regulation effect of bone morphogenetic protein 4 on cholinergic-expression for neurons of embryo rats / 解放军医学杂志

Objective To Observe the adjusting effect of bone morphogenetic protein 4 (BMP4) on cholinergic-expression in embryo rats' neurons. Method Neurons isolated from hippocampus and cerebral cortex of 15-20 days gestational age SD rat's brain were cultivated in a DMEM/F12 medium containing Ara-C and identified with morphological characters and microtubulin associated protein 2 (MAP2 ) immunocytochemical test. The medium was replaced with BMP4 96 hours after Ara-C adding, and cultivated another 10 days. The expression of choline acetyltransferase (ChAT) of the adjusted cells was observed by immunocytochemical staining and FITC-labeled cholinergic nerve cells Flow Cytometry. Results It has been showed that cultured neurons accorded with the typical morphological characters and the immunologic markers of neurons; the percentage of ChAT-positive cells in BMP4 group was prominently higher than that in control group(13.3?1.37% vs 6.44?0.81%, P

Changes of renal peripheral benzodiazepine receptor in the stress/anxiety response

Peripheral benzodiazepine receptor(PBR) has been identified in various peripheral tissues including kidney. The physiological and pharmacological functions of PBR are still uncertain, although it has been suggested that these are associated with the regulation of stress/anxiety response. Diazepam progeny, which were exposed to diazepam perinatally, was reported to be an animal model of chronic anxiety. However, PBR in the diazepam progenies are not known yet. In the present study, therefore, we examined the changes of PBR in the stress/anxiety response. Dams of rats were given injection of diazepam or vehicle during puerperium. Diazepam progenies showed increased level of anxiety on the performance of elevated plus maze, and increased Bmax of PBR. Saturation experiments followed by scatchard analysis of the results showed that the increase in the density of PBR and the affinity of the PBR remained unchanged. Forced swim stress increased anxiety on the plus maze in both groups of rats. In contrast to control, diazepam progenies did not show further upregulation of renal PBR immediately after swimming stress, but still higher than control. From the above results, it may be concluded that upregulation of renal PBR is associated with chronic anxiety as well as stress-induced response.