Agrimoniin is a dual inhibitor of AKT and ERK pathways that inhibit pancreatic cancer cell proliferation.

Molecular-targeted therapy has shown its effectiveness in pancreatic cancer, while single-targeted drug often cannot provide long-term benefit because of drug resistance. Fortunately, multitarget combination therapy can reverse drug resistance and achieve better efficacy. The typical treatment characteristics of traditional Chinese medicine monomer on tumor are multiple targets, with small side effects, low toxicity, and so forth. Agrimoniin has been reported to be effective on some cancers, while the mechanism still needs to be clarified. In this study, we used 5-ethynyl-2'-deoxyuridine, cell counting kit-8, flow cytometry, and western blot experiments to confirm that agrimoniin can significantly inhibit the proliferation of pancreatic cancer cell PANC-1 by inducing apoptosis and cell cycle arrest. In addition, by using SC79, LY294002 (the agonist or inhibitor of AKT pathway), and U0126 (the inhibitor of ERK pathway), we found that agrimoniin inhibited cell proliferation by simultaneously inhibiting AKT and ERK pathways. Moreover, agrimoniin could significantly increase the inhibitory effect of LY294002 and U0126 on pancreatic cancer cells. Meanwhile, in vivo experiments also supported the above results. In general, agrimoniin is a double-target inhibitor of AKT and ERK pathways in pancreatic cancer cells; it is expected to be used as a resistance reversal agent of targeted drugs or a synergistic drug of the inhibitor of AKT pathway or ERK pathway.

Inhibition of glutamine transporter ASCT2 mitigates bleomycin-induced pulmonary fibrosis in mice.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) represents a fatal pulmonary disease. Its mechanisms remain unclear and effective therapies are urgently needed. Glutaminolysis is involved in IPF pathology, but little is known about the role of ASCT2 responsible for cellular uptake of glutamine in IPF. We investigated the role of ASCT2 and its therapeutic implication in IPF through knockdown of ASCT2 in mice. METHODS: Mouse IPF model was established through a single intratracheal administration of bleomycin, and lentivirus-coated ASCT2 siRNA was administrated into mice via caudal vein for knockdown of ASCT2. Mouse blood and lung tissues were collected for biochemical, histological, and molecular examinations. RESULTS: ASCT2 siRNA significantly lowered ASCT2 expression in mouse lung tissues. Knockdown of ASCT2 reduced pulmonary levels of glutamic acid, α-ketoglutarate, glutathione and ATP, mitigated pulmonary histological injury, and reduced serum concentrations of pulmonary injury parameters including SP-A, SP-D, KL-6 and CCL18 in IPF mice. Moreover, serum levels of fibrotic parameters HA, LN, PC-III and IV-C were lowered by ASCT2 depletion. Collagen production and pulmonary hydroxyproline levels were also decreased by ASCT2 siRNA in IPF mice, which was concomitant with downregulation of α-smooth muscle actin, collagen type Iα1 and transforming growth factor-ß receptor II. Furthermore, ASCT2 deficiency downregulated the mRNA and protein expression of inflammatory cytokines IL-1ß and TNF-α as well as macrophage marker F4/80 in lung tissues of IPF mice. CONCLUSIONS: Inhibition of ASCT2 effectively mitigated pulmonary injury, fibrosis and inflammation in mice with bleomycin-induced IPF. ASCT2 could be a novel therapeutic target for treatment of IPF.

Vitamin D affects the neutrophil-to-lymphocyte ratio in patients with type 2 diabetes mellitus.

AIMS/INTRODUCTION: Chronic inflammation is an underlying feature of type 2 diabetes mellitus. Hypovitaminosis D is associated with type 2 diabetes mellitus, but whether it contributes to chronic inflammation is unclear. We examined the effects of vitamin D on various immune markers to evaluate its contribution to systemic inflammation in type 2 diabetes mellitus. MATERIALS AND METHODS: We retrospectively analyzed data from type 2 diabetes mellitus patients, people with prediabetes and control patients without diabetes (n = 9,746). Demographic and clinical variables were evaluated using descriptive statistics and generalized linear regression. A stratified analysis based on total serum vitamin D was also carried out. RESULTS: Neutrophil count was a significant predictor of 1,5-anhydroglucitol and glycated hemoglobin (HbA1c) in patients with prediabetes (1,5-anhydroglucitol: ß = -0.719, P < 0.001 and HbA1c: ß = -0.006, P = 0.002) and patients with diabetes (1,5-anhydroglucitol: ß = 0.207, P = 0.004 and HbA1c: ß = -0.067, P = 0.010). Lymphocyte count was a significant predictor of HbA1c in patients without diabetes (ß = 0.056, P < 0.001) and patients with prediabetes (ß = 0.038, P < 0.001). The neutrophil-to-lymphocyte ratio (NLR) was a significant predictor of HbA1c in patients without diabetes (ß = -0.001, P = 0.032). No immune markers differed significantly based on vitamin D level among patients without diabetes (P> 0.05 for all). Among patients with prediabetes, those who were vitamin D-deficient had the highest NLR (P = 0.040). Among patients with diabetes, those who were vitamin D-deficient had the highest neutrophil count (P = 0.001), lowest lymphocyte count (P = 0.016) and highest NLR (P < 0.001). CONCLUSIONS: The NLR is strongly influenced by serum vitamin D level. Given the high prevalence of hypovitaminosis D and elevated NLR among chronic disease patients and the elderly, our results suggest that clinical interpretation of NLR as a predictive marker of type 2 diabetes mellitus-related inflammation should consider vitamin D level, age and pre-existing morbidity.

MiR-135a Suppresses Calcification in Senescent VSMCs by Regulating KLF4/STAT3 Pathway.

Cellular function phenotype is regulated by various microRNAs (miRs), including miR-135a. However, how miR-135a is involved in the calcification in senescent vascular smooth muscle cells (VSMCs) is not clear yet. In the present study, we first identified the significantly altered miRNAs in VSMCs, then performed consecutive passage culture of VSMCs and analyzed the expression of miR- 135a and calcification genes in the senescent phase. Next, the effects of the miR-135a inhibition on calcification and calcification genes were analyzed. The luciferase assay was used to validate the target protein of miR-135a. The western blotting was used to determine the effects of miR-135a on Krüppel-like factor 4 (KLF4) and signal transducer and activator of transcription 3 protein (STAT3) expression, as well as the relationship between KLF4 and STAT3. Finally, the quantified cellular calcification was measured to examine the involvement of miR-135a, KLF4 and STAT3 in VSMCs calcification. Our results showed that miR-135a was significantly altered in VSMCs. Cell calcification and calcification genes were greatly altered by miR-135a inhibition. KLF4 was validated as the target RNA of miR-135a. Expression of KLF4 and STAT3 were both significantly decreased by over expressed miR-135a, while the inhibition of miR-135a and KLF4 siRNA both decreased the STAT3 protein levels. Moreover, the inhibition of miR-135a dramatically increased the calcium concentration, but co-treatment with KLF4 or STAT3 siRNA both decreased the calcium concentration. The present study identified miR-135a as a potential osteogenic differentiation suppressor in senescent VSMCs and revealed that KLF4/STAT3 pathway, at least partially, was involved in the mechanism.

Platycodin-D Induced Autophagy in Non-Small Cell Lung Cancer Cells via PI3K/Akt/mTOR and MAPK Signaling Pathways.

Platycodin-D (PD) is an effective triterpene saponin extracted from the root of Platycodon grandiflorum which has been used clinically to treat pulmonary diseases in traditional Chinese medicine. Recently, it has been reported that PD has anti-tumor effects in various cancer models through the induction of apoptosis. However, whether PD induces autophagy in both cell lines and its molecular mechanisms have not been elucidated. Here, our present study confirmed that PD induced autophagy in both NCI-H460 and A549 cells via up-regulating the expression levels of Atg-3, Atg-7 and Beclin-1. Meanwhile, PD contributed to the up-regulation of LC3-II at both protein and mRNA levels. Further detection of the PI3K/Akt/mTOR signaling pathway compared to LY294002 (PI3K kinase inhibitor), RAP (mTOR kinase inhibitor) and insulin (an activator of PI3K/Akt/mTOR signaling pathway) showed that PD induced autophagy through inhibiting the pathway at p-Akt (Ser473), p-p70S6K (Thr389) and p-4EBP1 (Thr37/46) in both cell lines. Moreover, the examination of MAPK signaling pathway showed that PD treatment increased the phosphorylation of JNK and p38 MAPK, while decreased the phosphorylation of Erk1/2 in both cell lines. Additionally, the effects assessed with a panel of pharmacologic inhibitors, including U0126 (Erk1/2 kinase inhibitor), SP600125 (JNK kinase inhibitor) and SB203580 (p38 MAPK kinase inhibitor) suggested that the activation of JNK and p38 MAPK participated in PD-induced autophagy. Taken together, these findings suggested that PD induced autophagy in NCI-H460 and A549 cells through inhibiting PI3K/Akt/mTOR signaling pathway and activating JNK and p38 MAPK signaling pathways. Therefore, PD may be an alternative compound for NSCLC therapy.

Up-regulated miR-93 contributes to coronary atherosclerosis pathogenesis through targeting ABCA1.

Atherosclerosis is a chronic inflammatory disease, starting with the accumulation of white blood cells and fatty materials in the arterial wall. ABCA1, a gene promotes phospholipid and cholesterol transfer from cells to poorly lapidated ApoA1, is considered to be related to the pathogenesis of coronary atherosclerosis. Meanwhile, disturbed miRNAs were reported to be related to coronary atherosclerosis. To understand the relationship between miRNA, ABCA1 and coronary atherosclerosis pathogenesis, we first screened the miRNAs that may directly target 3'UTR of ABCA1 and miR-33a was used as positive control. Through dual luciferase assay and western blot, we confirmed that miR-93 and miR-17 repress ABCA1 expression through directly targeting 3'UTR. The serum miR-33a, miR-93 and miR-17 levels in participants were detected by qRT-PCR and a significant reduction of miR-33a and miR-93 was found in the coronary patients. After statistical analysis we identified that a negative correlation was existed in the serum miR-93 and ABCA1 levels in coronary atherosclerosis patients. Meanwhile, our results indicate that the serum miR-93 positively correlates with the serum cholesterol level. This research may give insight into understanding of coronary atherosclerosis pathogenesis and create an opportunity to the diagnosis of coronary atherosclerosis.

The glucose RQ-feedback control leading to improved erythromycin production by a recombinant strain Saccharopolyspora erythraea ZL1004 and its scale-up to 372-m(3) fermenter.

In this paper, glucose respiratory quotient (RQ)-feedback control was developed for erythromycin production with a recombinant strain Saccharopolyspora erythraea ZL1004. RQ was confirmed to be an ideal online parameter for regulating glucose feed rate. Through feeding glucose to control RQ at 0.85 during 45-100 h and 0.95 during 100-185 h, erythromycin titer and erythromycin A concentration were reached 11.88 and 8.82 g l(-1) in 50 l fermenter, which were increased by 8.3 and 6.1 % as compared to that with glucose pH-feedback control, respectively. When glucose RQ-feedback control was scaled up to 372-m(3) fermenter, erythromycin titer and erythromycin A concentration at 155 h were reached 9.12 and 7.12 g l(-1), respectively, which were 10.5 and 9.4 % higher than that with the original technology (glucose pH-feedback control). To the best of our knowledge, this is the first report on the successful application of glucose RQ-feedback control in erythromycin production, especially in 372-m(3) fermenter.

[Molecular cloning of farnesyl pyrophosphate synthase from Alisma orientale (Sam.) Juzep. and its distribution pattern and bioinformatics analysis].

Triterpenes, which have large application potential in the treatment of cancer, are the main active components of genuine medicinal material Alisma orientale (Sam.) Juzep. Farnesyl pyrophosphate synthase (FPPS) is one of the important rate-limiting enzymes in the synthetic pathway of triterpenes. In this study the FPPS full length cDNA of the A. orientale, was cloned via homology-based cloning approach and rapid amplification of cDNA ends (RACE). The full length of the FPPS cDNA was 1 531 bp (accession no. HQ724508), which contained a full 1 032 bp ORF that encoded 343 amino acids. The deduced protein sequence exhibited five conserved motifs, two of which is riched of Asp (DDXXD). The result of real-time quantitative PCR (QRT-PCR) showed that FPPS gene was expressed in different organs of A. orientale. The expression increased from October to the first ten-day period of December, and then decreased. The FPPS gene expression was higher in leaves but lower in leafstalk, tuber and root. HPLC analysis of active components 23-acetyl-alismol B of A. orientale. during different periods indicated that its change trend should be consistent with FPPS gene expression. It can be primarily deduced that FPPS gene should be an important control point in the synthetic pathway of Alisma terpenes. This study may facilitate the quality of medicinal plants through gene engineering in the future.

T-cell vaccination leads to suppression of intrapancreatic Th17 cells through Stat3-mediated RORγt inhibition in autoimmune diabetes.

Immunization with inactivated autoreactive T cells is an effective therapeutic approach to ameliorating autoimmune diseases, while the underlying mechanisms that regulate autoreactive T cells are not completely understood. This study tested the hypothesis that T-cell vaccination (TCV) inhibits autoimmune diabetes in mice through the suppression of Th17 cells. The results showed that TCV treatment decreased hyperglycemia in type 1 diabetes (T1D) induced by multiple low-dose streptozotocin (MLD-STZ) as compared with the controls, preserved the number of healthy pancreatic islets and increased the production of insulin in the islets. Further study revealed that TCV significantly decreased the production of both interleukin (IL)-17 and IL-23 in intrapancreatic infiltrating lymphocytes (IPL) through marked inhibition of mRNA level of retinoic acid-related orphan receptor γt (RORγt) and signal transducer and activator of transcription 3 (Stat3) phosphorylation. The role of TCV-induced Th17 suppression was further validated in adoptive transfer experiments with polarized Th17 cells in sub-diabetogenic mice, which was similar to the effect of anti-IL-17 antibody treatment. Collectively our study shows that intrapancreatic Th17 cell suppression and healthy islet preservation play an important role in the treatment of T1D by TCV.

Rhein ameliorates fatty liver disease through negative energy balance, hepatic lipogenic regulation, and immunomodulation in diet-induced obese mice.

Nonalcoholic fatty liver disease (NAFLD) is associated with obesity, insulin resistance, and inflammatory disorders. In this study, we tested the effect of rhein, a lipophilic anthraquinone derived from a traditional Chinese herbal medicine Rheum palmatum L., on NAFLD-associated hepatic steatosis, insulin resistance, and the T helper (Th)1/Th2 cytokine imbalance in high-fat diet-induced obese (DIO) mice. We found that oral administration of rhein for 40 days significantly increased energy expenditure, reduced body weight, particularly body fat content, improved insulin resistance, and lowered circulating cholesterol levels in DIO mice without affecting food intake. Rhein treatment also reduced liver triglyceride levels, reversed hepatic steatosis, and normalized alanine aminotransferase (ALT) levels in these mice. Gene analysis and Western blot showed that rhein markedly suppressed the expression of the lipogenic enzyme sterol regulatory element-binding protein-1c (SREBP-1c) and its target genes in the liver. Luciferase reporter assay revealed that rhein suppressed the transcriptional activity of SREBP-1c through its upstream regulator, liver X receptor (LXR). This suggests that rhein exerts its effects by targeting LXR, which is also supported by its inability to reduce body weight in LXR knockout mice. Moreover, multiplex ELISA displayed a downregulated Th1 response after rhein treatment. Rhein shifted the Th1/Th2 responses by inhibiting T-box expressed in T-cells (T-bet) expression and enhancing GATA-binding protein-3 (GATA-3) expression through increased signal transducer and activator of transcription 6 (STAT6) phosphorylation. These data indicate that rhein ameliorated NAFLD and associated disorders through LXR-mediated negative energy balance, metabolic regulatory pathways, and immunomodulatory activities involved in hepatic steatosis. The combined effects of rhein to target hepatic metabolic and immune pathways may be beneficial for complex metabolic diseases such as NAFLD.

Liver X receptor (LXR) mediates negative regulation of mouse and human Th17 differentiation.

Th17 cells are a subset of CD4+ T cells with an important role in clearing certain bacterial and fungal pathogens. However, they have also been implicated in autoimmune diseases such as multiple sclerosis. Exposure of naive CD4+ T cells to IL-6 and TGF-ß leads to Th17 cell differentiation through a process in which many proteins have been implicated. We report here that ectopic expression of liver X receptor (LXR) inhibits Th17 polarization of mouse CD4+ T cells, while LXR deficiency promotes Th17 differentiation in vitro. LXR activation in mice ameliorated disease in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis, whereas LXR deficiency exacerbated disease. Further analysis revealed that Srebp-1, which is encoded by an LXR target gene, mediated the suppression of Th17 differentiation by binding to the E-box element on the Il17 promoter, physically interacting with aryl hydrocarbon receptor (Ahr) and inhibiting Ahr-controlled Il17 transcription. The putative active site (PAS) domain of Ahr and the N-terminal acidic region of Srebp-1 were essential for this interaction. Additional analyses suggested that similar LXR-dependent mechanisms were operational during human Th17 differentiation in vitro. This study reports what we believe to be a novel signaling pathway underlying LXR-mediated regulation of Th17 cell differentiation and autoimmunity.

[Cloning and distribution pattern of HMGR gene conserved fragment in Alisma orientale].

OBJECTIVE: To clone and study the distribution pattern of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene conserved fragment in Alisma orientale. METHODS: The HMGR conserved fragment in A. orientale was amplified by RT-PCR strategy with the total RNA of young leaves as the template. HMGR mRNA expression in different organs was detected by real-time quantitative PCR (QRT-PCR). RESULTS: The conserved fragment was 458 bp (accession NO. HQ913638). NCBI Blast sequence analysis showed the resulting protein had high homology to HMGR with 86.8% similarity to Artemisia annua, 88.2% to Bupleurum chinense, 88.2% to Eucommia ulmoides, and 85.5% to Salvia miltiorrhiza. The result of QRT-PCR showed that the HMGR gene was expressed in different organs (i. e. leaves, petioles, tubers, and roots) which was higher in leaves relative to other tissues. CONCLUSION: The HMGR gene conserved fragment from A. orientale was cloned and its distribution pattern was detected for the first time. This work provides a foundation for exploring the synthetic pathway and bioengineering of Alisma terpenes.