Correlation between the dynamic changes of γδT cells, Th17 cells, CD4+CD25+ regulatory T cells in peripheral blood and pharmacological interventions against bleomycin-induced pulmonary fibrosis progression in mice.

The involvement of γδT cells, Th17 cells, and CD4+CD25+ regulatory T cells (Tregs) is crucial in the progression of pulmonary fibrosis (PF), particularly in maintaining immune tolerance and homeostasis. However, the dynamics of these cells in relation to PF progression, especially under pharmacological interventions, remains poorly understood. This study aims to unravel the interplay between the dynamic changes of these cells and the effect of pharmacological agents in a mouse model of PF induced by intratracheal instillation of bleomycin. We analyzed changes in lung histology, lung index, hydroxyproline levels, and the proportions of γδT cells, Th17 cells, and Tregs on the 3rd, 14th, and 28th days following treatment with Neferine, Isoliensinine, Pirfenidone, and Prednisolone. Our results demonstrate that these drugs can partially or dynamically reverse weight loss, decrease lung index and hydroxyproline levels, and ameliorate lung histopathological damage. Additionally, they significantly modulated the abnormal changes in γδT, Th17, and Treg cell proportions. Notably, on day 3, the proportion of γδT cells increased in the Neferine and Prednisolone groups but decreased in the Isoliensinine and Pirfenidone groups, while the proportion of Th17 cells decreased across all treated groups. On day 14, the Neferine group showed an increase in all three cell types, whereas the Pirfenidone group exhibited a decrease. In the Isoliensinine group, γδT and Th17 cells increased, and in the Prednisolone group, only Tregs increased. By day 28, an increase in Th17 cell proportion was observed in all treatment groups, with a decrease in γδT cells noted in the Neferine group. These shifts in cell proportions are consistent with the pathogenesis changes induced by these anti-PF drugs, suggesting a correlation between cellular dynamics and pharmacological interventions in PF progression. Our findings imply potential strategies for assessing the efficacy and timing of anti-PF treatments based on these cellular changes.

Ethylene Acts as a Local and Systemic Signal to Mediate UV-B-Induced Nitrate Reallocation to Arabidopsis Leaves and Roots via Regulating the ERFs-NRT1.8 Signaling Module.

Nitrate is the preferred nitrogen source for plants and plays an important role in plant growth and development. Under various soil stresses, plants reallocate nitrate to roots to promote stress tolerance through the ethylene-ethylene response factors (ERFs)-nitrate transporter (NRT) signaling module. As a light signal, ultraviolet B (UV-B) also stimulates the production of ethylene. However, whether UV-B regulates nitrate reallocation in plants via ethylene remains unknown. Here, we found that UV-B-induced expression of ERF1B, ORA59, ERF104, and NRT1.8 in both Arabidopsis shoots and roots as well as nitrate reallocation from hypocotyls to leaves and roots were impaired in ethylene signaling mutants for Ethylene Insensitive2 (EIN2) and EIN3. UV-B-induced NRT1.8 expression and nitrate reallocation to leaves and roots were also inhibited in the triple mutants for ERF1B, ORA59, and ERF104. Deletion of NRT1.8 impaired UV-B-induced nitrate reallocation to both leaves and roots. Furthermore, UV-B promoted ethylene release in both shoots and roots by enhancing the gene expression and enzymatic activities of ethylene biosynthetic enzymes only in shoots. These results show that ethylene acts as a local and systemic signal to mediate UV-B-induced nitrate reallocation from Arabidopsis hypocotyls to both leaves and roots via regulating the gene expression of the ERFs-NRT1.8 signaling module.

CD4+CD25+ Tregs as dependent factor in the course of bleomycin-induced pulmonary fibrosis in mice.

The immune system is felt to play an essential role in pulmonary fibrosis (PF). CD4+CD25+ regulatory T cells (Tregs) are crucial in maintaining immune tolerance and immune homeostasis, but their role in the pathogenesis of PF is controversial and still unclear. We here explored the relationship between peripheral blood CD4+CD25+ Tregs and the course of bleomycin-induced PF in mice. Mouse PF models were established by intratracheal instillation of bleomycin. Lung histology, hydroxyproline, Th1/Th2 balanc, CD4+CD25+ Tregse were analyzed at the 3rd，7th，14th，21st and 28th days after instillation. CD4+CD25+ Tregs were also transferred into mice with or without PF by tail vein injection. The trend of CD4+CD25+ Tregs changes was increased firstly, decreased, increased again from 7th to 28th days after bleomycin instillation, which had great relevance with alveolitis and fibrosis scores. There also were high Th1 polarization index from 3rd to 14th days and high Th2 polarization index at 21st and 28th days after bleomycin treatment. CD4+CD25+ Tregs could promote the secretion of Th2 cytokines and inhibit the secretion of Th1 cytokines, allow the Th1/Th2 balance to Th2 direction in PF. Moreover, preventive adoptive transfer of CD4+CD25+ Tregs may ameliorate the process of PF, while acute adoptive transfer of CD4+CD25+ Tregs may aggravate the process of PF. These findings suggested that the dynamic changes of CD4+CD25+ Tregs as dependent factor might designate a different course of PF induced by bleomycin in mice, and might be a selected drug use indicator for therapy of PF.

A novel multiplex qPCR method for assessing the comparative lengths of telomeres.

BACKGROUND: The comparative length of telomeres is considered to be related to diseases such as cancer, aging, and cardiovascular diseases. qPCR is currently one of the main methods for detecting telomere length. However, due to the unique sequence of telomeres (highly repetitive six-base sequence), it is difficult to design primers and probes to expand and detect telomere and to put internal reference gene and telomere into the same tube for detection to reduce the possible inter-pore errors and improve amplification efficiency. Besides, the stability and accuracy of the test results are greatly affected by the difference between reference genes and telomere copy number. METHODS: In this study, the single-copy genes were replaced with high-copy genes (300 copies) as the internal control to reduce the copy number difference of the internal genes and telomere. In addition, a multiplex qPCR system was constructed to detect the telomeres and an internal reference gene product. We also detected the lengths of telomeres in the genomic DNA in immortalized cells (293T and Hela) from different generations of cells. RESULTS: We detected the comparative telomere lengths of 1500 random Chinese volunteers of different ages with the multiplex qPCR method; the result shows that the comparative length of telomeres is negatively related to age. In addition, we compared our qPCR detection method with a terminal restriction fragmentation (TRF) method. Both of them were highly consistent, indicating that the qPCR method was reliable. CONCLUSIONS: In conclusion, we developed a stable, convenient, and accurate comparative telomere length detection method.

A novel multiplex fluorescent competitive PCR for copy number variation detection.

The copy number variation (CNV) is an important genetic marker in cancer and other diseases. To detect CNVs of specific genetic loci, the multiplex ligation-dependent probe amplification (MLPA) is an appropriate approach, but the experimental optimization and probe synthesis are still great challenges. The multiplex competitive PCR is an alternative method for CNV detection. However, the construction of internal competitive template and establishment of a stable multiplex PCR system are the main limiting factors for this method. Here, we introduce a novel multiplex fluorescent competitive PCR (NMFC-PCR) for detecting CNVs. In this method, the blunt hairpin primers are used to rapidly establish a stable multiplex PCR system due to the reduction of non-specific amplification, and limited cycles' amplification is used to obtain the internal competitive template instead of artificial synthesis. With this method, we tested 21 clinical samples with potential LIM homeobox 1 (LHX1) or T-box 6 (TBX6) deletion. Every three segments located on the LHX1 and TBX6 were selected as the target regions, while two segments located on X-chromosome and five segments located on autosome were selected as the reference regions for detecting CNVs. The results showed that the gender information of 21 samples can be accurately inferred by the copy number ratio (CNR) of X-chromosomal reference region to autosomal reference region (X/A), and 2 samples had one copy of LHX1 and 9 samples had one copy of TBX6. To evaluate the accuracy of NMFC-PCR, 5 random samples with CNV were also detected by array-based comparative genomic hybridization (aCGH), and the results of aCGH were consistent with the NMFC-PCR results. To further assess the performance of NMFC-PCR, 60 normal samples were simultaneously tested. The results showed that the gender results were exactly the same as known information, and CNVs of LHX1 or TBX6 were not found. In conclusion, the method is a cheap, efficient, accurate, and convenient competitive PCR method for CNV detection.

TRPC3 overexpression and intervention in airway smooth muscle of ovalbumin-induced hyperresponsiveness and remodeling.

Transient receptor potential canonical channel 3 (TRPC3) proteins function as non-voltage-gated Ca2+ -permeable channels and play divergent roles in many processes of pathophysiology. The purpose of this study was to determine the relationship between TRPC3 expression and airway hyperresponsiveness and remodeling in ovalbumin-induced asthmatic Kunming mice. Mice were sensitized and challenged by ovalbumin to establish asthmatic model. Hematoxylin-eosin staining, hydroxyproline assay, and isometric tracheal ring force measurement were used to evaluate airway remodeling and hyperresponsiveness in asthmatic mice. Western blot was performed to detect the expression of TRPC3 proteins. MTT assay was used to measure the proliferation of airway smooth muscle cells. TRPC3 protein expression increased in airway smooth muscle of asthmatic mice. GdCl3 , a nonspecific TRPC blocker, attenuated the contractile force of airway smooth muscle. Fetal bovine serum stimulated airway smooth muscle cells proliferation and augmented TRPC3 protein expression. Both TRPC3 blockade by GdCl3 or specific TRPC3 antibodies and gene silencing by siRNA inhibited the proliferation of airway smooth muscle cells. In contrast, the current drugs treatment for asthma such as Dexamethasone and Aminophylline had no effects on TRPC3 protein overexpression. Therefore, TRPC3 protein overexpression may be involved in airway smooth muscle hyperresponsiveness and remodeling in asthmatic mice, providing evidence for a new direction of asthma pathogenesis research and a new target for drug intervention.

Deregulated expression of Cdc6 as BCR/ABL-dependent survival factor in chronic myeloid leukemia cells.

Chronic myeloid leukemia is characterized by the presence of the reciprocal translocation t(9;22) and the BCR/ABL oncogene. The BCR/ABL oncogene activates multiple signaling pathways and involves the dysregulation of oncogenes during the progression of chronic myeloid leukemia. The cell division cycle protein 6, an essential regulator of DNA replication, is elevated in some human cancer cells. However, the expression of cell division cycle protein 6 in chronic myeloid leukemia and the underlying regulatory mechanism remain to be elucidated. In this study, our data showed that cell division cycle protein 6 expression was significantly upregulated in primary chronic myeloid leukemia cells and the chronic myeloid leukemia cell line K562 cells, as compared to the normal bone marrow mononuclear cells. BCR/ABL kinase inhibitor STI571 or BCR/ABL small interfering RNA could significantly downregulate cell division cycle protein 6 messenger RNA expression in K562 cells. Moreover, phosphoinositide 3-kinase/AKT pathway inhibitor LY294002 and Janus kinase/signal transducer and activator of transcription pathway inhibitor AG490 could downregulate cell division cycle protein 6 expression in K562 cells, but not RAS/mitogen-activated protein kinase pathway inhibitor PD98059 had such effect. Cell division cycle protein 6 gene silencing by small interfering RNA effectively resulted in decrease of proliferation, increase of apoptosis, and arrest of cell cycle in K562 cells. These findings have demonstrated that cell division cycle protein 6 overexpression may contribute to the high proliferation and low apoptosis in chronic myeloid leukemia cells and can be regulated by BCR/ABL signal transduction through downstream phosphoinositide 3-kinase/Akt and Janus kinase/signal transducer and activator of transcription pathways, suggesting cell division cycle protein 6 as a potential therapeutic target in chronic myeloid leukemia.

Emerging Role of MicroRNAs and Long Noncoding RNAs in Healthy and Diseased Lung.

Pulmonary hypertension (PH) is a complex and multifactorial disease. An inability to fully unravel the molecular complexities has led to various clinical challenges in developing new therapies for this disease. Noncoding RNAs (ncRNAs) are RNA molecules with limited ability of coding proteins. The amount of ncRNAs is up to 98% of the whole genome's transcripts. Many ncRNAs with a regulatory function of genes have been identified to date and found to act at various steps along the protein biosynthetic process, which includes transcription, RNA maturation, translation, and protein degradation. These discoveries are fueling a new era in understanding the pathophysiology and therapeutic pathways of PH. In this chapter, we discuss the emerging role of noncoding RNAs in PH as well as other pulmonary diseases.

A novel three-round multiplex PCR for SNP genotyping with next generation sequencing.

Owing to the high throughput and low cost, next generation sequencing has attracted much attention for SNP genotyping application for researchers. Here, we introduce a new method based on three-round multiplex PCR to precisely genotype SNPs with next generation sequencing. This method can as much as possible consume the equivalent amount of each pair of specific primers to largely eliminate the amplification discrepancy between different loci. After the PCR amplification, the products can be directly subjected to next generation sequencing platform. We simultaneously amplified 37 SNP loci of 757 samples and sequenced all amplicons on ion torrent PGM platform; 90.5 % of the target SNP loci were accurately genotyped (at least 15×) and 90.4 % amplicons had uniform coverage with a variation less than 50-fold. Ligase detection reaction (LDR) was performed to genotype the 19 SNP loci (as part of the 37 SNP loci) with 91 samples randomly selected from the 757 samples, and 99.5 % genotyping data were consistent with the next generation sequencing results. Our results demonstrate that three-round PCR coupled with next generation sequencing is an efficient and economical genotyping approach. Graphical Abstract The schematic diagram of three-round PCR.

ceRNA in cancer: possible functions and clinical implications.

Competing endogenous RNAs (ceRNAs) are transcripts that can regulate each other at post-transcription level by competing for shared miRNAs. CeRNA networks link the function of protein-coding mRNAs with that of non-coding RNAs such as microRNA, long non-coding RNA, pseudogenic RNA and circular RNA. Given that any transcripts harbouring miRNA response element can theoretically function as ceRNAs, they may represent a widespread form of post-transcriptional regulation of gene expression in both physiology and pathology. CeRNA activity is influenced by multiple factors such as the abundance and subcellular localisation of ceRNA components, binding affinity of miRNAs to their sponges, RNA editing, RNA secondary structures and RNA-binding proteins. Aberrations in these factors may deregulate ceRNA networks and thus lead to human diseases including cancer. In this review, we introduce the mechanisms and molecular bases of ceRNA networks, discuss their roles in the pathogenesis of cancer as well as methods of predicting and validating ceRNA interplay. At last, we discuss the limitations of current ceRNA theory, propose possible directions and envision the possibilities of ceRNAs as diagnostic biomarkers or therapeutic targets.

Effects of neferine on Kv4.3 channels expressed in HEK293 cells and ex vivo electrophysiology of rabbit hearts.

AIM: Neferine is an isoquinoline alkaloid isolated from seed embryos of Nelumbo nucifera (Gaertn), which has a variety of biological activities. In this study we examined the effects of neferine on Kv4.3 channels, a major contributor to the transient outward current (I(to)) in rabbit heart, and on ex vivo electrophysiology of rabbit hearts. METHODS: Whole-cell Kv4.3 currents were recorded in HEK293 cells expressing human cardiac Kv4.3 channels using patch-clamp technique. Arterially perfused wedges of rabbit left ventricles (LV) were prepared, and transmembrane action potentials were simultaneously recorded from epicardial (Epi) and endocardial (Endo) sites with floating microelectrodes together with transmural electrocardiography (ECG). RESULTS: Neferine (0.1-100 µmol/L) dose-dependently and reversibly inhibited Kv4.3 currents (the IC50 value was 8.437 µmol/L, and the maximal inhibition at 100 µmol/L was 44.12%). Neferine (10 µmol/L) caused a positive shift of the steady-state activation curve of Kv4.3 currents, and a negative shift of the steady-state inactivation curve. Furthermore, neferine (10 µmol/L) accelerated the inactivation but not the activation of Kv4.3 currents, and markedly slowed the recovery of Kv4.3 currents from inactivation. Neferine-induced blocking of Kv4.3 currents was frequency-dependent. In arterially perfused wedges of rabbit LV, neferine (1, 3, and 10 µmol/L) dose-dependently prolonged the QT intervals and action potential durations (APD) at both Epi and Endo sites, and caused dramatic increase of APD10 at Epi sites. CONCLUSION: Neferine inhibits Kv4.3 channels likely by blocking the open state and inactivating state channels, which contributes to neferine-induced dramatic increase of APD10 at Epi sites of rabbit heart.

The relationship between frequency dependence of action potential duration and the expression of TRPC3 in rabbit ventricular myocardium.

BACKGROUND/AIMS: Cardiac action potential duration (APD) is regulated by heart rate, leading to the trans-membrane movement of inorganic ions. Whether the alteration of heart rate can affect the expression of transient receptor potential canonical channels (TRPCs), further studies should be made. We investigated the changes of APD at different stimulus frequencies and their influences on the expression of TRPCs in rabbit ventricular myocardium. METHODS: Monophasic action potential (MAP) was recorded by contact electrode technique in different programmed stimulus frequencies on rabbit ventricular epicardium in vivo, and the expression of TRPCs was detected using RT-PCR and Western blot. RESULTS: At the frequency range of 4.5-7.5 Hz, APD gradually shortened with the increase of stimulus frequency, showing the property of significant frequency dependence in rabbit ventricular myocardium in vivo. Compared with 4.5 Hz group, TRPC3 mRNA and protein expression increased in 6 Hz and 7.5 Hz groups by way of frequency dependence. Both amiodarone (AM) and neferine (Nef) could prolongate APD and showed characters of frequency independence at the designed frequency. In contrast with 4.5 Hz control group, it was Nef treatment group rather than AM treatment group that could obviously increase the expression of TRPC3 mRNA and protein. CONCLUSIONS: At the frequency range of 4.5-7.5 Hz, frequency-dependent shortening of APD was associated with the expression of TRPC3. AM and Nef exhibited frequency-independent lengthening of the APD. Nef may prolong APD via the increasement of TRPC3.

Efficacy and safety of adalimumab for the Crohn's disease: a systematic review and meta-analysis of published randomized placebo-controlled trials.

PURPOSE: The aim of this study was to evaluate the efficacy and safety of adalimumab (ADA) for Crohn's disease. METHODS: Electronic databases, including PubMed, Embase, the Cochrane Library, and the Science Citation Index, were searched to retrieve relevant trials. We estimated pooled estimates of the odds ratio (OR) and relevant 95% confidence interval (CI) using fixed effects model or random effects model as appropriate. RESULTS: Six randomized placebo-controlled studies met the selection criteria. Short-term clinical response/remission and long-term remission were better in the ADA groups than in the control groups (P < 0.05), both in anti-TNF-naive patients and in subjects who lost their response and/or became intolerant to infliximab (IFX). And ADA was also effective for patients who were previously treated with IFX, and its efficacy in infliximab-exposed patients was probably less than in infliximab-naive patients. In patients with active Crohn's disease (CD), ADA therapy was more effective than placebo for obtaining complete fistula closure. In comparison with placebo, ADA does not increase the risk of serious adverse events. CONCLUSIONS: ADA appears to be effective in achieving short-term clinical response/remission, long-term remission, and complete fistula healing in CD, including patients not manageable with IFX, and appears to have a favorable safety profile. A longer duration of follow-up and a larger number of patients are required to better assess the safety profile of ADA in CD.

Distinct activity of BK channel ß1-subunit in cerebral and pulmonary artery smooth muscle cells.

This study was designed to test a hypothesis that the functional activity of big-conductance, Ca(2+)-activated K(+) (BK) channels is different in cerebral and pulmonary artery smooth muscle cells (CASMCs and PASMCs). Using patch-clamp recordings, we found that the activity of whole cell and single BK channels were significantly higher in CASMCs than in PASMCs. The voltage and Ca(2+) sensitivity of BK channels were greater in CASMCs than in PASMCs. Targeted gene knockout of ß(1)-subunits significantly reduced BK currents in CASMCs but had no effect in PASMCs. Western blotting experiments revealed that BK channel α-subunit protein expression level was comparable in CASMCs and PASMCs; however, ß(1)-subunit protein expression level was higher in CASMCs than in PASMCs. Inhibition of BK channels by the specific blocker iberiotoxin enhanced norepinephrine-induced increase in intracellular calcium concentration in CASMCs but not in PASMCs. Systemic artery blood pressure was elevated in ß(1)(-/-) mice. In contrast, pulmonary artery blood pressure was normal in ß(1)(-/-) mice. These findings provide the first evidence that the activity of BK channels is higher in cerebral than in PASMCs. This heterogeneity is primarily determined by the differential ß(1)-subunit function and contributes to diverse cellular responses in these two distinct types of cells.

Upregulation of TRPC1 contributes to contractile function in isoproterenol-induced hypertrophic myocardium of rat.

AIMS: The transient receptor potential canonical channel 1 (TRPC1) is a crucial component of the stretch-activated ion channels (SACs). The objective of this research was to demonstrate the contribution of TRPC1 in maintaining cardiac contractile function in the hypertrophic myocardium. METHODS: Hypertrophic rat hearts were induced by injecting isoproterenol intraperitoneally, and the expressions of TRPC1/3/6 and Na(+)/Ca(2+) exchanger 1 (NCX1) proteins were analyzed by Western blot. The intracellular calcium images, the action potential of myocardium, the length-dependent contractile force of ventricle muscle and the cardiac output of isolated heart were investigated. RESULTS: The expression of TRPC1 was increased in the hypertrophic myocardium. After being stretched, the ascendant amplitude of the increase in the intracellular calcium ion concentration ([Ca(2+)]i) in the hypertrophic myocardium was higher than that in the normal myocardium. The increase of the APD50 and the amplitude of the membrane potential depolarization were more significant in the hypertrophic myocardium after the activation of SACs. When the heart preparations were perfused with Tyrode's solution, there was no difference in the cardiac systolic function between the cardiac hypertrophy group and the control group. Gadolinium, a SACs blocker, reduced the length-dependent contractile force and suppressed the ascending limb of the Frank-Starling curves in the hypertrophic heart. CONCLUSIONS: The upregulation of TRPC1 contributes to the contractile function in the hypertrophic myocardium by increasing [Ca(2+)]i through the SACs.

Quantitative analysis of multiple genes' expressions based on a novel competitive RT-PCR assay.

We established a novel gene expression analysis platform, Multiplex Competitive RT-PCR Using Fluorescent Universal Primers (MCF-PCR), to study multi-gene expression patterns simultaneously. This platform combines fluorescent universal primers, multiplex competitive RT-PCR, and capillary electrophoretic separation, which ensures MCF-PCR a reliable, medium-throughput, cost-effective technology for gene expression profiling. With cloned standard DNAs, the detection limits, precision, and sensitivity of MCF-PCR were evaluated and compared with that of the assay without adding competitive templates and real-time PCR, respectively. The results showed that detection limit was 3.125 × 10(3) to 3.2 × 10(6) copies, and 10 % copy differences between two samples can be detected by MCF-PCR. To validate MCF-PCR, we analyzed expression profile of five genes in interleukin (IL)-4/IL-13 pathway in peripheral blood of 20 healthy adults and 20 allergic dermatitis patients; three genes including IL-4, IL-13, and STAT6 were found differentially expressed in the two sample groups, which maybe key players in IL-4/IL-13 immunological signaling pathway and need further function analysis.

Benzylisoquinoline alkaloids inhibit lung fibroblast activation mainly via inhibiting TGF-ß1/Smads and ERK1/2 pathway proteins.

Backgrounds: Liensinine (Lien), Neferine (Nef), Isoliensinine (Iso) and Tetrandrine (Tet), benzylisoquinoline alkaloids (BIAs), have been shown inhibitory effects on pulmonary fibrosis (PF) through anti-inflammatory, anti-oxidative activities, inhibition of cytokines and NF-κB. Effects of other similar BIAs, Dauricine (Dau), Papaverine (Pap) and lotusine (Lot), on PF remain unclear. Here, we explored the effects of five bisbenzylisoquinoline (Lien, Nef, Iso, Tet and Dau) and two monobenzylisoquinoline (Pap, Lot) alkaloids on normal and PF fibroblasts. Methods: Primary normal and PF lung fibroblasts were cultured and treated with these alkaloids. Proliferation, activation, migration and apoptosis changes were detected by MTT, wound healing assay, flow cytometry. Protein level was analyzed by Western blot. Results: All BIAs inhibited proliferation of normal and PF lung fibroblasts induced by TGF-ß. α-SMA protein level in normal and PF lung fibroblasts decreased after Lien, Nef, Iso, Tet and Dau treatment. Pap and Lot had no influence on α-SMA expression. Dau showed the strongest inhibitory effects on proliferation and activation among alkaloids. The migration rates of normal and PF lung fibroblasts were inhibited by Lien, Nef, Iso, and Dau. Lien, Nef, Iso and Dau significantly promoted apoptosis, while Tet had no effect on apoptosis. Pap and Lot had no influence on activation, migration and apoptosis. Dau significantly inhibited Smad3/4 and p-ERK1/2 protein overexpression induced by TGF-ß1. Conclusions: Bisbenzylisoquinoline alkaloids had stronger effects on inhibiting lung fibroblasts than monobenzylisoquinoline alkaloids. Dau expressed the strongest inhibitory effects, which may be related to its inhibition of TGF-ß1/Smad3/4 and p-ERK1/2 pathway proteins.

Beneficial effects of THSG on acetic acid-induced experimental colitis: involvement of upregulation of PPAR-γ and inhibition of the Nf-Κb inflammatory pathway.

The polyphenolic compound 2,3,5,4'-tetrahydroxystilbene-2-O-beta-D-glucoside (THSG) has been shown to possess anti-inflammatory effects. Here, we examined the effects of THSG on experimental mice with colitis induced by acetic acid and whether the underlying mechanisms were associated with the PPAR-γ and NF-κB pathways. Mice were randomized into six equal groups: normal, colitis model, THSG (10, 30, 60 mg·kg(-1)) and mesalazine. The mice were administered 10, 30, 60 mg·kg(-1) THSG or 100 mg·kg-1 mesalazine or saline once daily by intragastric administration for 7 days after induction of colitis by acetic acid irrigation. THSG dramatically attenuated acetic acid-induced colon lesions, including reversing the body weight loss and improving histopathological changes. THSG apparently decreased the increase of malondialdehyde (MDA) which is a marker of lipid peroxidation. THSG appears to exert its beneficial effects on acetic acid-induced experimental colitis through upregulation of PPAR-γ mRNA and protein levels and inhibition of the NF-κB pathway, which in turn decreases the protein overexpression of the downstream inflammatory mediators TNF-α, IL-6 and COX-2. The effect of THSG 60 mg·kg(-1) on PPAR-γ mRNA expression was higher than that of mesalazine. THSG may thus be a promising new candidate or lead compound for the treatment of IBD.

Functional role of canonical transient receptor potential 1 and canonical transient receptor potential 3 in normal and asthmatic airway smooth muscle cells.

Canonical transient receptor potential (TRPC)-encoded nonselective cation channels (NSCCs) are crucial for many cellular responses in a variety of cells; however, their molecular expression and functional roles in airway smooth muscle cells (ASMCs) remain obscure. The objective of this study was to determine whether TRPC1 and TRPC3 molecules could be important molecular constituents of native NSCCs controlling the resting membrane potential (Vm) and [Ca(2+)](i) in freshly isolated normal and ovalbumin (OVA)-sensitized/-challenged mouse ASMCs. Western blotting, RT-PCR, single-channel recording, whole-cell current-clamp recording, and a fluorescence imaging system were used to determine TRPC expression, NSCC activity, resting Vm, and resting [Ca(2+)](i). Specific individual TRPC antibodies and siRNAs were applied to test their functional roles. TRPC1 and TRPC3 proteins and mRNAs were expressed in freshly isolated ASM tissues. TRPC3 antibodies blocked the activity of NSCCs and hyperpolarized the resting Vm in ASMCs, whereas TRPC1 antibodies had no effect. TRPC3, but not TRPC1 gene silencing, largely diminished NSCC activity, hyperpolarized the resting Vm, lowered the resting [Ca(2+)](i), and inhibited methacholine-induced increase in [Ca(2+)](i). In OVA-sensitized/-challenged ASMCs, NSCC activity was greatly augmented, resting Vm was depolarized, and TRPC3 protein expression was increased. TRPC1 and TRPC3 antibodies blocked the increased activity of NSCCs and membrane depolarization in OVA-sensitized/-challenged cells. TRPC3 is an important molecular component of native NSCCs contributing to the resting Vm and [Ca(2+)](i) in normal ASMCs, as well as membrane depolarization and hyperresponsiveness in OVA-sensitized/-challenged cells, whereas TRPC1-encoded NSCCs are only activated in OVA-sensitized/-challenged airway myocytes.

[Development, optimization and application of the expression analysis platform based on multiplex quantitative RT-PCR using fluorescent universal primers].

A multiplex quantitative RT-PCR technology with a universal fluorescent primer was established. This technology employs a chimeric-primer-induced-universal-primer amplification method that ensures target genes amplified in a constant ratio. This technique was cost-effective, moderate-throughput, and reliable in quantification of gene expression. It is complementary to cDNA chip, which has low quantitative accuracy , and Real-time quantitative PCR with low throughput, through improving the entire process of expression profiling analysis. Eleven genes within a QTL segment regulating mouse puberty onset on chromosome X were investigated to construct and optimize the method. The sensitivity of detection (102 copies) was determined, the concentration ratio of universal primer and chimeric forward primers (1:1) was optimized, and the accuracy and repeatability were validated. The method of Touchdown PCR with addition of universal primers significantly improved amplification of genes expressed in low abundance. After testing the expression profile of 11 genes in hypothalamus and testis in two mouse strains C3H/HeJ and C57BL/6J at the age of 15 d, one gene named PHF6 was found differentially expressed for further function analysis.