Importance of Gedunin in Antagonizing Rheumatoid Arthritis via Activating the Nrf2/ARE Signaling.

Objective: This study assessed the anti-arthritic effect and protection of Gedunin (GDN) on joint tissues and revealed the possible mechanism in suppressing rheumatoid arthritis (RA). Methods: LPS-induced macrophages and TNF-α-stimulated synovial fibroblasts (MH7A) or IL-1ß-stimulated primary rheumatoid arthritis synovial fibroblasts (RASFs) were used to evaluate the antiinflammatory effect of GDN. In addition, CIA-induced arthritis was employed here to evaluate the anti-arthritic effect. MTT and BRDU assays were utilized to evaluate the cell viability and proliferation, Q-PCR was conducted to detect the mRNA expression of cytokines, FACS was adopted to monitor ROS production, while western blotting (WB) and siRNA interference were applied in confirming the anti-arthritic effects of GDN via the Nrf2 signaling. Results. In vitro, cell viability was inhibited in macrophages and MH7A cells, but not in RASFs; but the proliferation of RASFs was significantly suppressed in time- and dose-dependent manners. GDN suppressed cytokine levels in LPS-stimulated macrophages and TNF-α-stimulated MH7A cells or RASFs. GDN suppressed ROS expression. Furthermore, GDN treatment notably dose-dependently decreased the mRNA and protein expression of iNOS in LPS-induced macrophages. sip62 interference results showed that GDN cause the less expression of HO-1 and Keap1 and also fail to inhibit cytokines after sip62 interference. In vivo, GDN effectively inhibited paw swelling, arthritis score, and arthritis incidence and cytokines. Conclusions: Our study suggested that GDN exhibited strong antagonistic effect on arthritis both in vitro and in vivo via activation of Nrf2 signaling. Our work will provide a promising therapeutic strategy for RA.

Neobaicalein Inhibits Th17 Cell Differentiation Resulting in Recovery of Th17/Treg Ratio through Blocking STAT3 Signaling Activation.

Huangqin is the dried root of Scutellaria baicalensis Georgi, which has been widely utilized for heat-clearing (Qingre) and dewetting (Zaoshi), heat-killed (Xiehuo) and detoxifying (Jiedu) in the concept of Traditional Chinese Medicine and is used for treating inflammation and cancer in clinical formulas. Neobaicalein (NEO) is of flavonoid isolated from Huangqin and has been reported to possess prominent anti-inflammatory effects in published work. Th17/Treg balance shift to Th17 cells is an essential reason for autoimmune inflammatory diseases. However, the role NEO plays in Th17 and Treg and the underlying mechanism has not been elucidated yet. Network pharmacology-based study revealed that NEO predominantly regulated IL-17 signaling pathway. Moreover, our result shown that NEO (3-30 µmol/L) down-regulated Th17 differentiation and cellular supernatant and intracellular IL-17A level and tumor necrosis factor α production in a concentration-dependent manner. The further mechanism research revealed that NEO also specifically inhibited phosphorylation of STAT3(Tyr725) and STAT4 (Y693) without influence on activation of STAT5 and STAT6 in splenocytes. Immunofluorescence results illuminated that NEO effectively blocked STAT3 translocated into nucleus. Interestingly, NEO at appreciated dose could only inhibit Th17 cell differentiation and have no effect on Treg differentiation. The present study revealed that NEO effectively inhibited Th17 cell differentiation through specifically blocking the activation of STAT3 signaling without inactivation of STAT5 and STAT6. Additional inhibitory effect on activation of STAT4 by NEO also suggested the potential for antagonism against Th1 differentiation. All work suggested that NEO may be a potential candidate for immunoregulation and treating autoimmune inflammatory diseases through inhibiting immune cell viability and T cell differentiation.

Dendrobium mixture attenuates renal damage in rats with diabetic nephropathy by inhibiting the PI3K/Akt/mTOR pathway.

Dendrobium mixture (DMix) is a Traditional Chinese Medicine widely used for preventing and treating diabetic nephropathy (DN). Autophagy contributes to DN development and progression. The present study aimed to investigate the mechanism underlying the protective effects of DMix on the kidneys of rats with DN and to determine whether this involves autophagy. Herein, a high­sugar and high­fat diet, combined with the intra­abdominal injection of low­dose streptozocin, was used to induce DN in 40 Sprague­Dawley male rats. In total, 10 additional rats were used as controls. The rats with DN were then randomly divided into three groups and treated with DMix, gliquidone or saline via gastric administration for 8 weeks. Body weight, kidney weight, kidney index, fasting blood glucose (FBG), blood lipid, hemoglobin A1c (HbA1c), insulin, blood urea nitrogen and serum creatinine levels, as well as the 24­h urinary albumin excretion rate (UAER) were measured. H&E, Periodic Acid­Schiff and Masson staining were used to examine the renal pathology. The mRNA and protein expression levels of LC3 and Beclin­1 in renal tissues were measured using reverse transcription­quantitative PCR and immunohistochemistry, respectively. Western blotting was conducted to measure the protein expression levels of PI3K, phosphorylated (p)­PI3K, Akt, p­Akt, mTOR, p­mTOR, LC3 and Beclin­1 in renal tissues. It was found that DMix significantly reduced the FBG, blood lipids, HbA1c and insulin levels, kidney weight, kidney index and UAER in rats with DN, as well as improved renal function. Rats with DN showed notable glomerular hypertrophy, an increase in mesangial matrix content and renal interstitial fibrosis. Moreover, DMix notably reduced kidney damage. The results demonstrated that DMix inhibited the phosphorylation of PI3K, Akt and mTOR in the kidney tissues of rats with DN, and increased the protein and mRNA expression levels of LC3 and Beclin­1. Therefore, it was suggested that DMix has protective effects on the kidney of rats with DN, which may be associated with the inhibition of the PI3K/Akt/mTOR signaling pathway and activation of renal autophagy by this traditional medicine.

A novel tri-culture model for neuroinflammation.

Neuroinflammation is believed to play a primary role in the pathogenesis of most neurodegenerative diseases including Alzheimer's disease, Parkinson's disease and schizophrenia. Currently, suitable in vitro neuroinflammation models for studying cellular interactions and inflammatory mechanisms at the neurovascular unit are still scarce. In this study, we established an experimentally flexible tri-culture neuroinflammation model combining murine microglial cells (N11), mouse neuroblastoma Nuro2A cell lines and brain microvascular endothelial MVEC(B3) cells in a transwell co-culture system stimulated with lipopolysaccharides. Neuroinflammation was induced in this tri-culture model as manifested by activated N11 cells via toll-like receptor 4, resulting in increased release of proinflammatory mediators (nitric oxide, interleukin-6 and tumour necrosis factor-α) through the activation of nuclear factor-κB signalling pathway. The released inflammatory cytokines from N11 in turn, damaged the tight junction in microvascular endothelial MVEC(B3) cells, increased permeability of endothelial barrier, and induced tau phosphorylation and up-regulated caspase-3 expression in mouse neuroblastoma Nuro2A cell lines, leading to neuroinflammation injury. In summary, this tri-culture inflammation model mimics the microenvironment, the cellular crosstalk and the molecular events that take place during neuroinflammation. It provides a robust in vitro model for studying neuroinflammation mechanisms and screening for potential therapeutics to treat various neurodegenerative diseases.

PLCD1 Suppressed Cellular Proliferation, Invasion, and Migration via Inhibition of Wnt/ß-Catenin Signaling Pathway in Esophageal Squamous Cell Carcinoma.

BACKGROUND: Phospholipase C delta 1 (PLCD1) has been found to be abnormally expressed in various cancers. However, the potential roles of PLCD1 in esophageal squamous cell carcinoma (ESCC) are still unknown. METHODS: Western blot and qPCR were used to explore PLCD1 expression in various ESCC cells. MTT, colony formation assays, wound-healing assay, and transwell cell invasion assay were used to examine the cell viability in vitro. Western blot, qPCR, and luciferase assays were used to investigate the effects of PLCD1 on Wnt/ß-catenin signaling pathway. The xenograft models in nude mice were established to explore the roles of PLCD1 in vivo. RESULTS: We found that the expression of PLCD1 in ESCC cells was significantly downregulated than that in normal esophageal epithelial cells. In addition, upregulation of PLCD1 decreased the capacity of TE-1 and EC18 cells in proliferation, invasion, and migration. Then, the expression of ß-catenin/p-ß-catenin, C-myc, cyclin D1, MMP9, and MMP7 was investigated. PLCD1 activity was found to be negatively associated with the expression of ß-catenin, C-myc, cyclin D1, MMP9, and MMP7. Finally, the activity of PLCD1 in inhibiting ESCC proliferation in vivo was validated. CONCLUSION: The inhibitory effects of PLCD1 on the proliferation, invasion, and migration of TE-1 and EC18 cells might be associated with inhibition of Wnt/ß-catenin signaling pathway. PLCD1 played a key role in inhibiting ESCC carcinogenesis and progression in patients with ESCC.

NADH protect against radiation enteritis by enhancing autophagy and inhibiting inflammation through PI3K/AKT pathway.

Radiotherapy is an important method for cancer treatment but it has serious side-effects at high doses. One of the greatest challenges in radiotherapy is that radiation affects both healthy tissue and cancer tissues. For abdominal or pelvic lesions, the bowel is the most easily injured by irradiation. Radiation may cause radiation enteritis, intestinal inflammatory infiltration, or intestinal perforation. Coenzyme NADH involves in energy metabolism and transportation of nucleic acid, proteins and carbohydrates. In our study, NADH was used to protect the intestinal wall from irradiation injury in IEC-6 normal intestinal epithelial cells. By flow cytometry, we found that NADH can inhibit the cell death and the producing of reactive oxygen species (ROS). The immunofluorescence assay showed that cell autophagy was increased in the NADH group. Western blot data indicated that NADH promoted the microtubule associated protein 1A/1B-light chain 3(LC3)-I to LC3II and the expression of IL-1ß and TNFα decreased in a dose dependent manner. Interestingly, a specific PI3K/AKT inhibitor (3MA) decreased the expression of inflammatory factors. In the animal experiment, after 12 Gy radiation, there were less TNFα and more LC3II in the RT+NADH group than that of RT group. Compared with the mock, there was no significant damage in the NADH group. Thus, our study provides the evidence that NADH may protect against radiation enteritis by suppressing inflammation and enhancing autophagy through PI3K/AKT pathway in normal intestinal cells.

EPB41L3 is a potential tumor suppressor gene and prognostic indicator in esophageal squamous cell carcinoma.

Although there have been reports about the role of erythrocyte membrane protein band 4.1 like 3 (EPB41L3) in several types of cancer, primarily in non-small-cell lung carcinoma, the molecular function and modulatory mechanisms of EPB41L3 remain unclear. In specific, the functional and clinical significance of EPB41L3 in esophageal squamous cell carcinoma (ESCC) has not been explored to date. In the present study, reduced EPB41L3 expression was demonstrated in ESCC cell lines and tissues, which was due to its high methylation rate. Ectopic expression of EPB41L3 in ESCC cells inhibited cell proliferation in vivo and in vitro. In addition, EPB41L3 overexpression induced apoptosis and G2/M cell cycle arrest by activating Caspase-3/8/9 and Cyclin-dependent kinase 1/Cyclin B1 signaling, respectively. Notably, patients with higher EPB41L3 expression had markedly higher overall survival rates compared with patients with lower EPB41L3 expression. In summary, the present results suggest that EPB41L3 may be a tumor suppressor gene in ESCC development, representing a potential therapeutic target and a prognostic indicator for ESCC.

A novel three-dimensional tumorsphere culture system for the efficient and low-cost enrichment of cancer stem cells with natural polymers.

Cancer stem cells (CSCs) are considered to serve a key role in tumor progression, recurrence and metastasis. Tumorsphere culture is the most important method for enriching CSCs and is widely used in basic research and drug screening. However, the traditional suspension cell culture system has several disadvantages, including low efficiency, high cost and difficult procedure, making it difficult to produce tumorspheres on a large scale. In the present study, two biomaterials, methylcellulose (MC) and gellan gum (GG), were used to construct a novel culture system based on the traditional system. Subsequently, the characteristics of the novel three-dimensional (3D) culture system were evaluated, the design scheme was optimized, and the morphological and biological features of the tumorspheres cultured in this 3D system were compared with the traditional system. The results revealed that the tumorspheres cultured in the novel 3D system presented a higher seeding density and improved morphology, while maintaining stem-like properties. This evidence suggests that a simple, efficient and low-cost culture system that produces tumorspheres on a large scale was successfully constructed, which can be widely used in various aspects of stem cell research.

Suppression of Lipogenesis via Reactive Oxygen Species-AMPK Signaling for Treating Malignant and Proliferative Diseases.

AIMS: Systemic diseases often have common characteristics. The aim of this study was to investigate the feasibility of targeting common pathological metabolism to inhibit the progression of malignant and proliferative diseases. RESULTS: Gefitinib-resistant (G-R) nonsmall-cell lung cancer (NSCLC) and rheumatoid arthritis (RA) were studied as conditions representative of malignant and proliferative diseases, respectively. Strong lipogenic activity and high expression of sterol regulatory element-binding protein 1 (SREBP1) were found in both G-R NSCLC cells and synovial fibroblasts from RA patients (RASFs). Berberine (BBR), an effective suppressor of SREBP1 and lipogenesis regulated through reactive oxygen species (ROS)/AMPK pathway, selectively inhibited the growth of G-R NSCLC cells and RASFs but not that of normal cells. It effectively caused mitochondrial dysfunction, activated ROS/AMPK pathway, and finally suppressed cellular lipogenesis and cell proliferation. Addition of ROS blocker, AMPK inhibitor, and palmitic acid significantly reduced the effect of BBR. In an in vivo study, treatment of BBR led to significant inhibition of mouse tumor xenograft growth and remarkably slowed down the development of adjuvant-induced arthritis in rats. Innovation and Conclusion: Targeting ROS/AMPK/lipogenesis signaling pathway selectively inhibited the growth of G-R NSCLC cells and the progress of RASFs in vitro and in vivo, which provides a new avenue for treating malignancies and proliferative diseases. Antioxid. Redox Signal. 28, 339-357.

Growth suppressive effect of pegylated arginase in malignant pleural mesothelioma xenografts.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a difficult-to-treat global disease. Pegylated arginase (BCT-100) has recently shown anti-tumor effects in hepatocellular carcinoma, acute myeloid leukemia and melanoma. This study aims to investigate the effects of PEG-BCT-100 in MPM. METHODS: A panel of 5 mesothelioma cell lines (H28, 211H, H226, H2052 and H2452) was used to study the in vitro effects of BCT-100 by crystal violet staining. The in vivo effects of BCT-100 were studied using 211H and H226 nude mice xenografts. Protein expression (argininosuccinate synthetase, ornithine transcarbamylase, cleaved PARP, cleaved caspase 3, cyclins (A2, D3, E1 and H), CDK4 and Ki67) and arginine concentration were evaluated by Western blot and ELISA respectively. Cellular localization of BCT-100 was detected by immunohistochemistry and immunoflorescence. TUNEL assay was used to identify cellular apoptotic events. RESULTS: Argininosuccinate synthetase was expressed in H28, H226, and H2452 cells as well as 211H and H266 xenografts. Ornithine transcarbamylase was undetectable in all cell lines and xenograft models. BCT-100 reduced in vitro cell viability (IC50 values at 13-24 mU/ml, 72 h) across different cell lines and suppressed tumor growth in both 211H and H226 xenograft models. BCT-100 (60 mg/kg) significantly suppressed tumor growth (p < 0.01) with prolonged median survival (p < 0.01) in both xenograft models. Combining BCT-100 with pemetrexed or cisplatin conferred no additional benefits over single agents. Serum and intratumoral arginine levels were effectively decreased by BCT-100, associated with cytosolic accumulation of BCT-100 within tumor cells. Apoptosis (PARP cleavage in 211H xenografts; Bcl-2 downregulation, and cleavage of PARP and caspase 3 in H226 xenografts; positive TUNEL staining in both) and G1 arrest (downregulation of cyclin A2, D3, E1 and CDK4 in 211H xenografts; suppression of cyclin A2, E1, H and CDK4 in H226 xenografts) were evident with BCT-100 treatment. Furthermore, proliferative factor Ki67 was downregulated in BCT-100 treatments arms. CONCLUSIONS: BCT-100 suppressed tumor growth with prolonged median survival partially mediated by intratumoral arginine depletion resulting in apoptosis and G1 arrest in mesothelioma xenograft models. The findings provide scientific evidence to support further clinical development of BCT-100 in treatment of MPM.

MiR-15a suppresses hepatocarcinoma cell migration and invasion by directly targeting cMyb.

PURPOSE: This study aimed to determine the function of miR-15a in HCC, and identify cMyb as a target of miR-15a. METHODS: RNA expression was evaluated by quantitative real-time PCR (qRT-PCR). The effects of miR-15a or cMyb on HCC cells were evaluated by transwell migration assay and western blot analysis. CMyb, the predicted target, has been frequently verified by luciferase assay. RESULTS: MiR-15a was markedly downregulated in sphere culture HCC cells by qRT-PCR. CMyb was predicted to be a potential target of miR-15a using bioinformatics analysis. This prediction has been frequently verified by luciferase assay and western blot. A positive correlation between cMyb and the migration ability of HCC cells was demonstrated by transwell assays. MiR-15a mimic suppressed cMyb expression to weaken HCC cell migration ability. On the other hand, miR-15a inhibitor upregulated cMyb and induced HCC cell migration. CONCLUSION: MiR-15a could suppress HCC progression through the repression of cMyb, making miR-15a a potential therapeutic target.

Role of axl in preeclamptic EPCs functions.

Axl encodes the tyrosine-protein kinase receptor, participating in the proliferation and migration of many cells. This study examined the role of Axl in functions of endothelial progenitor cells (EPCs). Axl was detected by RT-PCR and Western blotting in both placentas and EPCs from normal pregnancy and preeclampsia patients. The Axl inhibitor, BMS777-607, was used to inhibit the Axl signalling pathway in EPCs. Cell proliferation, differentiation, migration and adhesion were measured by CCK-8 assay, cell differentiation assay, Transwell assay, and cell adhesion assay, respectively. Results showed the expression levels of Axl mRNA and protein were significantly higher in both placentas and EPCs from preeclampsia patients than from normal pregnancy (P<0.05). After treatment with BMS777-607, proliferation, differentiation, migration and adhesion capability of EPCs were all significantly decreased. Our study suggests Axl may play a role in the function of EPCs, thereby involving in the pathogenesis of preeclampsia.

Epb41l3 suppresses esophageal squamous cell carcinoma invasion and inhibits MMP2 and MMP9 expression.

EPB41L3 may play a role as a metastasis suppressor by supporting regular arrangements of actin stress fibres and alleviating the increase in cell motility associated with enhanced metastatic potential. Downregulation of epb41l3 has been observed in many cancers, but the role of this gene in esophageal squamous cell carcinoma (ESCC) remains unclear. Our study aimed to determine the effect of epb41l3 on ESCC cell migration and invasion. We investigated epb41l3 protein expression in tumour and non-tumour tissues by immunohistochemical staining. Expression in the non-neoplastic human esophageal cell line Het-1a and four ESCC cell lines - Kyse150, Kyse510, Kyse450 and Caes17 - was assessed by quantitative Polymerase Chain Reaction (qPCR) and Western blotting. Furthermore, an EPB41L3 overexpression plasmid and EPB41L3-specific small interfering RNA were used to upregulate EPB41L3 expression in Kyse150 cells and to downregulate EPB41L3 expression in Kyse450 cells, respectively. Cell migration and invasion were evaluated by wound healing and transwell assays, respectively. The expression levels of p-AKT, matrix metalloproteinase (MMP)2 and MMP9 were evaluated. Expression of epb41l3 was significantly lower in tumour tissues than in non-tumour tissues and in ESCC cell lines compared with the Het-1a cell line. Kyse450 and Caes17 cells exhibited higher expression of epb41l3 than Kyse150 and Kyse510 cells. Overexpressing epb41l3 decreased Kyse150 cell migration and invasion, whereas EPB41L3-specific small interfering RNA silencing increased these functions in Kyse450 cells. Furthermore, overexpressing epb41l3 led to downregulation of MMP2 and MMP9 in Kyse150 and Kyse510 cells. Our findings reveal that EPB41L3 suppresses tumour cell invasion and inhibits MMP2 and MMP9 expression in ESCC cells.

Discovery of Anti-inflammatory Ingredients in Chinese Herbal Formula Kouyanqing Granule based on Relevance Analysis between Chemical Characters and Biological Effects.

Kouyanqing Granule (KYQG) is a traditional Chinese herbal formula composed of Flos lonicerae (FL), Radix scrophulariae (RS), Radix ophiopogonis (RO), Radix asparagi (RA), and Radix et rhizoma glycyrrhizae (RG). In contrast with the typical method of separating and then biologicalily testing the components individually, this study was designed to establish an approach in order to define the core bioactive ingredients of the anti-inflammatory effects of KYQG based on the relevance analysis between chemical characters and biological effects. Eleven KYQG samples with different ingredients were prepared by changing the ratios of the 5 herbs. Thirty-eight ingredients in KYQG were identified using Ultra-fast liquid chromatography-Diode array detector-Quadrupole-Time-of-flight-Tandem mass spectrometry (UFLC-DAD-Q-TOF-MS/MS) technology. Human oral keratinocytes (HOK) were cultured for 24 hours with 5% of Cigarette smoke extract (CSE) to induce inflammation stress. Interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumour necrosis factor-α (TNF-α) were evaluated after treatment with the eleven KYQG samples. Grey relational analysis(GRA), Pearson's correlations (PCC), and partial least-squares (PLS) were utilized to evaluate the contribution of each ingredient. The results indicated that KYQG significantly reduced interleukin-1ß, interleukin-6, interleukin-8, and tumour necrosis factor-α levels, in which lysine, γ-aminobutyric acid, chelidonic acid, tyrosine, harpagide, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isoquercitrin, luteolin-7-o-glucoside, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, angoroside C, harpagoside, cinnamic acid, and ruscogenin play a vital role.

The laxative effect of emodin is attributable to increased aquaporin 3 expression in the colon of mice and HT-29 cells.

Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is a stimulant laxative and used to treat constipation. Aquaporin 3 (AQP3) plays an important role in regulating water transfer in the colon. In the study, we investigated whether the laxative effect of emodin is associated with the regulation of AQP3 in the colon. Our results showed that treatment with emodin increased the fecal water content in the colon of mice and evaluation index of defecation in a dose-dependent manner. More interestingly, emodin significantly increased the AQP3 protein and mRNA expression both in the colon of mice and in human intestinal epithelial cells (HT-29). Mechanistically, emodin obviously up-regulated the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A catalytic subunits α (PKA C-α) and phosphorylated cAMP response element-binding protein (p-CREB Ser133) expression in HT-29 cells. These results suggest that the laxative effect of emodin is associated with the increased expression of AQP3 by up-regulating PKA/p-CREB signal pathway.

Effects of four antitussives on airway neurogenic inflammation in a guinea pig model of chronic cough induced by cigarette smoke exposure.

OBJECTIVE: The effects of four antitussives, including codeine phosphate (CP), moguisteine, levodropropizine (LVDP) and naringin, on airway neurogenic inflammation and enhanced cough were investigated in guinea pig model of chronic cough. METHODS: Guinea pigs were exposed to CS for 8 weeks. At the 7th and 8th week, the animals were treated with vehicle, CP (4.8 mg/kg), moguisteine (24 mg/kg), LVDP (14 mg/kg) and naringin (18.4 mg/kg) respectively. Then the cough and the time-enhanced pause area under the curve (Penh-AUC) during capsaicin challenge were recorded. The substance P (SP) content, NK-1 receptor expression and neutral endopeptidase (NEP) activity in lung were determined. RESULTS: Chronic CS exposure induced a bi-phase time course of cough responsiveness to capsaicin. Eight weeks of CS exposure significantly enhanced the airway neurogenic inflammation and cough response in guinea pigs. Two weeks of treatment with CP, moguisteine, LVDP or naringin effectively attenuated the chronic CS-exposure enhanced cough. Only naringin exerted significant effect on inhibiting Penh-AUC, SP content and NK-1 receptor expression, as well as preventing the declining of NEP activity in lung. CONCLUSIONS: Chronic CS-exposed guinea pig is suitable for studying chronic pathological cough, in which naringin is effective on inhibiting both airway neurogenic inflammation and enhanced cough.

Protective effects of naringin against paraquat-induced acute lung injury and pulmonary fibrosis in mice.

The present study evaluates protective effects of naringin against paraquat (PQ)-induced acute lung injury (ALI) and pulmonary fibrosis in mice. Survival probability against PQ intoxication was tested by a single intraperitoneal injection of PQ. Results showed that survival rates of mice exposed to PQ only (50 mg/kg within 7 days) were much lower than that in mice daily treatment with NAC or naringin. Moreover, protection against PQ-induced ALI was tested by daily pretreatment mice with saline, NAC or naringin for 3 days before PQ (30 mg/kg, i.p.). Results showed that increase in leukocytes infiltration and overexpressions of TNF-α and TGF-ß1 caused by 8h of PQ exposure were dose-dependently ameliorated by naringin. Furthermore, protection against PQ-induced pulmonary fibrosis was tested by pretreatment mice with PQ (20 mg/kg, i.p.), and then daily administration with saline, NAC or naringin for prolonged 21 days. Results showed that naringin of 60 and 120 mg/kg significantly reduced PQ-induced upregulations of TNF-α, TGF-ß1, MMP-9 and TIMP-1, levels of pulmonary malonaldehyde and hydroxyproline, as well as pulmonary fibrosis deposition, while increased activities of SOD, GSH-Px and HO-1. These results indicated that naringin had effective protection against PQ-induced ALI and pulmonary fibrosis.

[Effect of anti-HPV16 E6-ribozyme on cell proliferation and invasiveness of cervical carcinoma cell line CaSKi].

OBJECTIVE: To investigate the effects of anti-HPV16-ribozyme on the cell proliferation and invasiveness of cultured cervical cancer cell line CaSKi. METHODS: CaSKi cells were transfected with anti-HPV16 E6-ribozyme and empty eukaryotic expression plasmids via lipofectin and designated as CaSKi-R and CaSKi-P cells respectively. The growth rate, cell colony-forming ability on soft agar, invasiveness and tumorigenicity of CaSKi-R, CaSKi-P, and CaSKi cells were studied using corresponding methods. The expressions of cox-2 and vascular endothelial growth factor (VEGF) mRNA of the 3 cell strains were determined with one-step reverse transcriptional PCR (RT-PCR), and immunocytochemistry was employed for detecting the expressions of COX-2 and VEGF antigens. RESULTS: No distinct differences in the growth rate, colony-forming ability on soft agar, cell invasiveness and tumorigenicity were observed between CaSKi and CaSKi-P cells, whereas by comparison, CaSKi-R cells exhibited decreased growth rate, colony-forming ability, the cell invasiveness and tumorgenicity, with also lowered expression levels of cox-2 and VEGF mRNA as shown by RT-PCR analysis. Expressions of COX-2 and VEGF antigens were detected in all the 3 cell strains immunocytochemically, but compared with CaSKi and CaSKi-P cells, the antigen expressions in CaSKi-R cells were significantly weaker. CONCLUSION: Anti-HPV16 E6-ribozyme may partially inhibit the proliferation and reduce the invasiveness of CaSKi cells possibly through decreasing cox-2 and VEGF expressions, which are the important agents for tumor invasion.

[Effects of anti-HPV16 E6-ribozyme on invasiveness of cervical carcinoma cell line CaSKi and vascular endothelial growth factor expression].

OBJECTIVE: To investigate the changes in the invasiveness of cervical cancer cell line CaSKi and the expression of vascular endothelial growth factor (VEGF) in response to treatment with anti-HPV16 E6-ribozyme, which plays a important role in the malignant phenotype and conversion of cervical cancer cells. METHODS: By means of lipofectin transfection, anti- HPV16 E6-ribozyme and empty eukaryotic expression plasmids were respectively transfected into CaSKi cell line (designated as CaSKi-R and CaSKi-P respectively). CaSKi, CaSKi-R and CaSKi-P cells were observed for their cell growth curves, clone forming ability on soft agar and tumorigenicity in nude mice. One-step reverse transcriptional PCR (RT-PCR) was employed to examine the expression of VEGF. RESULTS: No significant differences were found in the growth rate, clone forming ability and tumorigenicity between CaSKi and CaSKi-P cells. In contrast, CaSKi-R exhibited obviously decreased growth rate, clone forming ability and tumorigenicity (P<0.05). RT-PCR analysis showed that the expression levels of VEGF mRNA in CaSKi-R cells were lower than those in CaSKi-P and CaSKi cells. CONCLUSION: Anti-HPV16 E6-ribozyme may reduce the proliferative ability and invasiveness of cervical cancer cell line CaSKi, possibly through decreasing VEGF expression in CaSKi cells.