Ginsenoside Rg1 ameliorates hypoxia-induced pulmonary arterial hypertension by inhibiting endothelial-to-mesenchymal transition and inflammation by regulating CCN1.

Pulmonary arterial hypertension (PAH) is a chronic obstructive disease characterized by vascular remodeling. Studies have confirmed that ginsenoside Rg1 can improve pulmonary hypertension to a certain extent, but the potential mechanism by which it improves hypoxia-induced PAH remains unclear. The aim of this study was to investigate the therapeutic effect of ginsenoside Rg1 on hypoxia-induced PAH. The results showed that hypoxia promoted inflammation, EndMT, and vascular remodeling, which were accompanied by decreased CCN1 levels and increased p-NFκB p65, TGF-ß1, and p-Smad 2/3 levels. Treatment with ginsenoside Rg1, recombinant CCN1, BAY-11-7082, and SB-431542 could prevent hypoxia-induced vascular remodeling, reduce the expression of the hypoxia-induced inflammatory cytokines TNF-α and IL-1ß, inhibit the expression of the mesenchymal markers α-SMA and Vimentin and restore the expression of the endothelial markers CD31 and VE-cadherin to improve hypoxia-induced EndMT, which may be associated with the upregulation of CCN1 protein expression and downregulation of p-NFκB p65, TGF-ß1, and p-Smad 2/3 in rats and cells. siRNA CCN1 transfection increased the expression of p-NFκB p65, TGF-ß1, and p-Smad 2/3 and accelerated the occurrence and development of inflammation and EndMT after hypoxia. In summary, our study indicated that hypoxia-induced EndMT and inflammation play a role in hypoxic pulmonary hypertension (HPH). Ginsenoside Rg1 treatment could reverse hypoxia-induced EndMT and inflammation by regulating CCN1 and has potential value in the prevention and treatment of HPH.

Astragaloside IV improves pulmonary arterial hypertension by increasing the expression of CCN1 and activating the ERK1/2 pathway.

The aim of the present study was to investigate the underlying mechanism of AS-IV and CCN1 in PAH and to evaluate whether the protective effect of AS-IV against PAH is associated with CCN1 and its related signalling pathway. In vivo, male SD rats were intraperitoneally injected with monocrotaline (MCT, 60 mg/kg) or exposed to hypoxia (10% oxygen) and gavaged with AS-IV (20, 40 and 80 mg/kg/day) to create a PAH model. In vitro, human pulmonary artery endothelial cells (hPAECs) were exposed to hypoxia (3% oxygen) or monocrotaline pyrrole (MCTP, 60 µg/mL) and treated with AS-IV (10, 20 and 40 µM), EGF (10 nM, ERK agonist), small interfering CCN1 (CCN1 siRNA) and recombinant CCN1 protein (rCCN1, 100 ng/mL). We identified the differences in the expression of genes in the lung tissues of PAH rats by proteomics. At the same time, we dynamically detected the expression of CCN1 by Western blot both in vivo and in vitro. The Western blot experimental results showed that the expression of CCN1 increased in the early stage of PAH and decreased in the advanced stage of PAH. The results showed that compared with the control group, MCT- and hypoxia-induced increased the hemodynamic parameters and apoptosis. AS-IV can improve PAH, as characterized by decreased hemodynamic parameters, vascular wall area ratio (WA%), vascular wall thickness ratio (WT%) and α-SMA expression and inhibition of cell apoptosis. Moreover, the improvement of PAH by AS-IV was accompanied by increased CCN1 expression, which activated the ERK1/2 signalling pathway. Meanwhile, CCN1 and p-ERK1/2 were inhibited by siCCN1 and promoted by rCCN1. EGF not only activated the ERK1/2 signalling pathway but also induced the expression of CCN1. In conclusion, AS-IV improves PAH by increasing the expression of CCN1 and activating the ERK1/2 signalling pathway. The results of our study provide a theoretical basis for additional study on the protective effect of AS-IV against PAH.

Ginsenoside Rg1 attenuates mechanical stress-induced cardiac injury via calcium sensing receptor-related pathway.

BACKGROUND: Ginsenoside Rg1 (Rg1) has been well documented to be effective against various cardiovascular disease. The aim of this study is to evaluate the effect of Rg1 on mechanical stress-induced cardiac injury and its possible mechanism with a focus on the calcium sensing receptor (CaSR) signaling pathway. METHODS: Mechanical stress was implemented on rats through abdominal aortic constriction (AAC) procedure and on cardiomyocytes and cardiac fibroblasts by mechanical stretching with Bioflex Collagen I plates. The effects of Rg1 on cell hypertrophy, fibrosis, cardiac function, [Ca2+]i, and the expression of CaSR and calcineurin (CaN) were assayed both on rat and cellular level. RESULTS: Rg1 alleviated cardiac hypertrophy and fibrosis, and improved cardiac decompensation induced by AAC in rat myocardial tissue and cultured cardiomyocytes and cardiac fibroblasts. Importantly, Rg1 treatment inhibited CaSR expression and increase of [Ca2+]i, which similar to the CaSR inhibitor NPS2143. In addition, Rg1 treatment inhibited CaN and TGF-ß1 pathways activation. Mechanistic analysis showed that the CaSR agonist GdCl3 could not further increase the [Ca2+]i and CaN pathway related protein expression induced by mechanical stretching in cultured cardiomyocytes. CsA, an inhibitor of CaN, inhibited cardiac hypertrophy, cardiac fibrosis, [Ca2+]i and CaN signaling but had no effect on CaSR expression. CONCLUSION: The activation of CaN pathway and the increase of [Ca2+]i mediated by CaSR are involved in cardiac hypertrophy and fibrosis, that may be the target of cardioprotection of Rg1 against myocardial injury.

Protective Effect of Astragaloside IV on High Glucose-Induced Endothelial Dysfunction via Inhibition of P2X7R Dependent P38 MAPK Signaling Pathway.

Vascular endothelial dysfunction is associated with increased mortality in patients with diabetes. Astragaloside IV (As-IV) is a bioactive saponin with therapeutic potential as an anti-inflammatory and antiendothelial dysfunction. However, the underlying mechanism for how As-IV ameliorated endothelial dysfunction is still unclear. Therefore, in this study, we examined the protective effect of As-IV against endothelial dysfunction and explored potential molecular biology mechanism. In vivo, rats were intraperitoneally injected with streptozotocin (STZ) at a dose of 65 mg/kg body weight to establish a diabetic model. In vitro studies, rat aortic endothelial cells (RAOEC) were pretreated with As-IV, SB203580 (p38 MAPK inhibitor) for 2 h prior to the addition of high glucose (33 mM glucose). Our findings indicated that As-IV improved impaired endothelium-dependent relaxation and increased the levels of endothelial NO synthase (eNOS) and nitric oxide (NO) both in vivo and in vitro. Besides, As-IV treatment inhibited the elevated inflammation and oxidative stress in diabetic model both in vivo and in vitro. Moreover, As-IV administration reversed the upregulated expression of P2X7R and p-p38 MAPK in vivo and in vitro. Additionally, the effects of both P2X7R siRNA and SB203580 on endothelial cells were similar to As-IV. Collectively, our study demonstrated that As-IV rescued endothelial dysfunction induced by high glucose via inhibition of P2X7R dependent p38 MAPK signaling pathway. This provides a theoretical basis for the further study of the vascular endothelial protective effects of As-IV.

Astragaloside IV attenuates myocardial ischemia/reperfusion injury in rats via inhibition of calcium-sensing receptor-mediated apoptotic signaling pathways.

Astragaloside IV (AsIV) is an active saponin extracted from Astragalus membranaceus, which has shown cardioprotective effects in a number of experimental animals. In this study we investigated the molecular mechanisms by which AsIV attenuated the myocardial ischemia reperfusion (MI/R)-induced injury in vitro and in vivo by focusing on calcium-sensing receptor (CaSR) and extracellular signal-regulated kinase 1/2 (ERK1/2). Rat neonatal cardiac myocytes were subjected to a hypoxia/reoxygenation (H/R) procedure in vitro, which significantly decreased the cell viability, increased lactate dehydrogenase (LDH) release, induced cardiomyocyte apoptosis, and increased [Ca2+]i. H/R also increased the expression of CaSR and decreased ERK1/2 phosphorylation levels in H/R-exposed myocytes. Pretreatment with AsIV (60 µmol/L) significantly improved the cell viability and decreased LDH release, attenuated myocyte apoptosis, decreased [Ca2+]i and CaSR expression, and increased the ERK1/2 phosphorylation levels. The protective effects of AsIV against H/R injury were partially inhibited by co-treatment with a CaSR agonist, gadolinium chloride (GdCl3) or with a specific ERK1/2 inhibitor U0126. For in vivo studies, a rat MI/R model was established. Pre-administration of AsIV (80 mg/kg every day, ig) significantly decreased the myocardium infarct size, creatine kinase-MB (CK-MB) production, serum cardiac troponin (cTnI) levels, and cardiomyocyte apoptosis in the rats with MI/R injury. The therapeutic effects of AsIV were associated with the downregulation of CaSR expression and upregulation of ERK1/2 phosphorylation in myocardial tissues. In summary, astragaloside IV attenuates myocardial I/R injury via inhibition of CaSR/ERK1/2 and the related apoptotic signaling pathways.

[Effect of Combined Intervention of Electroacupuncture and Astragaloside IV on Myocardial Hypertrophy and TGF-ß 1/Smad Signaling in Rats with Myocardial Fibrosis].

OBJECTIVE: To observe the effect of combined intervention of electroacupuncture (EA) and astragaloside IV(ASIV) on cardiac hypertrophy and transforming growth factor ß 1 (TGF-ß 1)/Smad signaling in isoproterenol (ISO) induced cardiac hypertrophy rats, so as to investigate its underlying mechanisms in improving myocardial fibrosis. METHODS: A total of 50 SD rats were randomly divided into 5 groups: normal control, model (ISO), Propranolol (PRO)，ASIV and EA＋ASIV groups (n＝10 in each group). The myocardial fibrosis model was established by intraperitoneal injection (i.p.) of ISO (10 mg·kg－1·d－1), once daily for 30 days. Rats of the control group were given normal saline (i.p.), those of the PRO group given with PRO (40 mg·kg－1·d－1, gavage), and those of the ASIV and EA＋ASIV groups were treated by gavage of ASIV (40 mg·kg－1·d－1), once daily for 30 days. EA (20 Hz, 6 V) was applied to bilateral "Neiguan" (PC 6) for 10 min, once every day for 30 d. The heart mass index (HMI, whole heart weight/body weight) and left ventricular (LV) mass index (LVMI, weight of the LV/body weight) were calculated to assess the state of cardiac hypertrophy. The enzyme linked immunosorbent assay (ELISA) was used to determine the levels of procollagen I carboxy-terminal propeptide (PICP，a marker of extracellular matrix remodeling) and carboxyterminal telopeptide of type I collagen (ICTP, a metabolite of type I collagen) in serum, and Western blot was used to test protein contents of TGF- ß 1, Smad 2 / 3, Smad 4, Smad 7 in the left ventricle tissue of the heart. RESULTS: After modeling, the HMI and LVMI, serum PICP and ICTP contents and the expression levels of myocardial TGF-ß 1, Smad 2/3 and Smad 4 proteins were significantly increased in the model (ISO) group (P<0.05), suggesting a deposition of collagen and cardiac hypertrophy, and were considerably decreased in PRO, ASIV and EA＋ASIV groups after the intervention (P<0.05). The expression level of myocardial Smad 7 protein was significantly lower in the model group than in the normal control group (P<0.05), and significantly up-regulated in PRO, ASIV and EA＋ASIV groups (P<0.05). Sirius Red staining of the left ventricular myocardium showed a dense deposition of collagen and a severer myocardial fibrosis in the model group, and a relatively lighter fibrosis in the PRO, ASIV and EA＋ASIV groups. The therapeutic effects of EA＋ASIV were comparable to those of PRO, and were significantly superior to those of ASIV in down-regulating HMI, serum ICTP, and myocardial Smad 2/3 and Smad 4 expression and up-regulating Smad 7 protein (P<0.05). There were no significant differences among the PRO, ASIV and EA＋ASIV groups in LVMI, PICP and TGF-ß 1 levels, and between the PRO and EA＋ ASIV groups in HMI, ICTP, Smad 2/3, Smad 4 and Smad 7 levels (P> 0.05). CONCLUSIONS: EA stimulation of PC 6 combined with ASIV can relieve cardiac hypertrophy and myocardial fibrosis in rats, which may be associated with its effects in regulating myocardial TGF-ß 1/Smad signaling pathway.

Potential Signaling Pathways Involved in the Clinical Application of Oxymatrine.

Oxymatrine, an alkaloid component extracted from the roots of Sophora species, has been shown to have antiinflammatory, antifibrosis, and antitumor effects and the ability to protect against myocardial damage, etc. The potential signaling pathways involved in the clinical application of oxymatrine might include the TGF-ß/Smad, toll-like receptor 4/nuclear factor kappa-light-chain-enhancer of activated B cells, toll-like receptor9/TRAF6, Janus kinase/signal transduction and activator of transcription, phosphatidylinositol-3 kinase/Akt, delta-opioid receptor-arrestinl-Bcl-2, CD40, epidermal growth factor receptor, nuclear factor erythroid-2-related factor 2/heme oxygenase-1 signaling pathways, and dimethylarginine dimethylaminohydrolase/asymmetric dimethylarginine metabolism pathway. In this review, we summarize the recent investigations of the signaling pathways related to oxymatrine to provide clues and references for further studies on its clinical application. Copyright © 2016 John Wiley & Sons, Ltd.

Vibration exercise decreases insulin resistance and modulates the insulin signaling pathway in a type 2 diabetic rat model.

Vibration exercise (VE) is a new type of physical training, but little is known about its effects on insulin resistance at the molecular level. A Sprague-Dawley rat model of type 2 diabetes with insulin resistance was induced with a high-fat diet and low-dose streptozotocin. Animals were then subjected to 8 wk of VE consisting of placing the rats on a vibration stand bracket (8 cm × 8 cm × 20 cm) with a maximum vertical vibration displacement of 52 mm for 15 min twice a day, 6 d each week. Various metabolic markers and the phosphorylation and expression of proteins within the PI3K/AKT insulin signaling pathway were assessed. The high-fat diet and low-dose streptozotocin increased food intake, body weight, and levels of blood glucose, triglycerides, total cholesterol, and free fatty acids, while Kitt rate, 2-deoxyglucose uptake, and glycogen levels were decreased. These effects were ameliorated in animals receiving VE. VE treatment activated the PI3K/AKT insulin-signaling pathway, and also increased the expression of GLUT4. In conclusion, VE improved the metabolic issues associated with the diabetic state by suppressing the reduction of IRS1, AKT2, and GLUT4 in the diabetic condition, indicating that VE could be used as a therapeutic intervention for insulin resistance and type 2 diabetes.

Zhuanggu Jianxi Decoction () limits interleukin-1 ß-induced degeneration chondrocytes via the caveolin-p38 MAPK signal pathway.

OBJECTIVE: To evaluate the effect of Zhuanggu Jianxi Decoction (, ZGJXD) on interleukin-1 ß (IL-1 ß)-induced degeneration of chondrocytes (CDs) as well as the activation of caveolin-p38 mitogen-activated protein kinase (MAPK) signal pathway, investigating the possible molecular mechanism that ZGJXD treats osteoarthritis. METHODS: Serum pharmacology was applied in the present study, where ZGJXD was orally administrated to New Zealand rabbits and then ZGJXD containing serum (ZGJXD-S) was collected for following in vitro experiments. CDs were isolated aseptically from New Zealand rabbits and then cultured in vitro. Upon IL-1 ß stimulation, the degeneration of CDs was verified by inverted microscope, toluidine blue stain and type II collagen immunocytochemistry. After IL-1 ß-stimulated CDs were intervened with blank control serum, ZGJXD-S, together with or without SB203580 (a specific inhibitor of p38 MAPK) for 48 h, caveolin-1 protein expression and the phosphorylation level of p38 were determined by Western blotting, and the mRNA expression of IL-1 ß, tumor necrosis factor α (TNF-α), matrix metalloproteinase 3 (MMP-3) and MMP-13 were examined by real-time polymerase chain reaction. RESULTS: IL-1 ß stimulation induced degeneration of CDs, increased caveolin-1 expression and p38 phosphorylation, up-regulated the mRNA level of IL-1 ß, TNF-α, MMP-3 and MMP-13. However, the IL-1 ß-induced activation of caveolin-p38 signaling and alteration in the expression of p38 downstream target genes were suppressed by ZGJXD-S and/or SB203580 in CDs. CONCLUSION: ZGJXD can prevent CDs degeneration via inhibition of caveolin-p38 MAPK signal pathway, which might be one of the mechanisms that ZGJXD treats osteoarthritis.

[Effect of naringin of Drynaria Rhizome, a Chinese medical component of Zhuanggu Jianxi Recipe containing serum on caveolin-p38MAPK signal pathway in IL-1ß induced rabbit degenerated chondrocytes].

OBJECTIVE: To observe the effect of naringin of Drynaria Rhizome, a Chinese medical component of Zhuanggu Jianxi Recipe (ZJR) containing serum on caveolin-p38MAPK signal factors (such as caveolin-1, p-p38, p-ATF-2, IL-1ß, and TNF-α) in IL-1ß induced rabbit degenerated chondrocytes, and further to explore its mechanism for protecting articular cartilages. METHODS: Naringin of Drynaria Rhizome was obtained and analyzed by HPLC-TOF/MS. Four weeks old New Zealand rabbits were killed and their bilateral knee joints were isolated aseptically. CDs were isolated and then cultured in vitro. The second generation of CDs were used for later experiment. The effect of naringin on CDs proliferation was detected by MTT assay. The effect of naringin on the expression of IL-1ß-induced collagen II in CDs was detected by immunohistochemical method. The effect of naringin on caveolin-1, p-p38, and p-ATF-2 protein in IL-1ß-induced CDs was detected by Western blot. The effect of naringin on mRNA expression of IL-1ß and TNF-α in IL-1ß-induced CDs was detected by RT-PCR. RESULTS: The appearance time of naringin in flow graphs of naringin standard solution and ZJR containing serum was 23.5 min, and the molecular weight ranged between 581.0 and 581.5 m/z. Naringin could promote the proliferation of CDs, and inhibit the effect of IL-1ß on collagen II in CDs. Compared with the model group, naringin could reduce the expression of caveolin-1, p-p38, p-ATF-2, IL-1ß, and TNF-α in IL-1ß induced CDs (P < 0.05), which was approximate to the level of the normal group. CONCLUSIONS: Naringin could not only promote the proliferation of CDs, but also protect IL-1ß-induced CDs. Its mechanism might be associated with decreasing the expression of caveolin-1, p-p38, and p-ATF-2 proteins, inhibiting caveolin-p38MAPK signal pathway, and further reducing mRNA expression of IL-1ß and TNF-α in the downstream of caveolin-p38MAPK signal pathway.

Astragaloside IV attenuates inflammatory cytokines by inhibiting TLR4/NF-кB signaling pathway in isoproterenol-induced myocardial hypertrophy.

ETHNOPHARMACOLOGICAL RELEVANCE: Astragaloside IV (As IV) is one of the main effective components isolated from the traditional Chinese medical herb Astragalus membranaceus. The protective effect of Astragalus membranaceus on myocardial hypertrophy has been extensively proved. To test the hypothesis that Astragaloside IV can ameliorate the myocardial hypertrophy and inflammatory effect induced by ß-adrenergic hyperactivity, we carried out in vivo and in vitro experiments. MATERIAL AND METHODS: In in vivo study, the isoproterenol (Iso) (5 mg kg(-1) d(-1)) was used as a model of myocardial hypertrophy by intraperitoneal injection. SD rats were randomly assigned to following six groups: A: the control; B: Iso group; C: Iso plus As IV 20 mg kg(-1) d(-1); D: Iso plus As IV 40 mg kg(-1) d(-1); E: Iso plus As IV 80 mg kg(-1) d(-1); F: Iso plus Propranolol 40 mg kg(-1) d(-1). In in vitro study, cultured neonatal rat cardiomyocytes were pretreated with As IV (3, 10, 30 µ mol L(-1)), Propranolol (2 µ mol L(-1)) and BAY11-7082 (5 µ mol L(-1)) for 30 min, and then incubated with Iso (10 µ mol L(-1)) for 48 h. For the rats in each group, the heart mass index (HMI) and the left ventricular mass index (LVMI) were measured. To measure the transverse diameter of left ventricular myocardial cells (TDM), the hematoxylin-eosin (HE) staining method was applied. In addition, the volume and the total protein content of cardiomyocytes were measured, the mRNA expression of ANP and TLR4 were quantified by RT-PCR, the protein expression of TLR4, IκBα and p65 were quantified by Western blot, and the level of TNF-α and IL-6 were measured by ELISA. RESULTS: In vivo: Comparing the Iso group to the control, the HMI, LVMI, TDM were significantly increased; the protein expression of TLR4 and p65 were increased, while the IκBα were decreased; the expression of ANP, TLR4 mRNA, and TNF-α, IL-6 in serum were significantly increased. These changes could be partly prevented by As IV and Pro. In vitro: the over-expression of the cell size, total protein content could remarkably down-regulated by As IV and Pro, and the results of RT-PCR, Western blot and ELISA were similar to those of in vivo. CONCLUSIONS: The results of these studies indicate that Astragaloside IV has good protective effect on myocardial hypertrophy induced by isoproterenol. More specifically, the cardioprotection is related to inhibiting the TLR4/NF-кB signaling pathway and the attenuating inflammatory effect.

[Effects of kappa-opioid receptor stimulation on high glucose induced myocardial hypertrophy of neonatal rats].

OBJECTIVE: To observe the effects of kappa-opioid receptor stimulation on high glucose induced myocardial hypertrophy of neonatal rats. METHODS: Using cultured myocardial cells as a model, the protein content was assayed with Lowrys method. The cardiomyocytes volumes were measured by computer photograph analysis system. The level of p-ERK44/42 was determined by Western blot. RESULTS: Compared with the control, U50488H significantly inhibited the protein content and volumes of cultured hypertrophic myocardial cells induced by high glucose. Meanwhile the role of ERK was important. CONCLUSION: The stimulation of kappa-opioid can inhibit myocardial hypertrophy induced by high glucose, which is possibly via attenuating p-ERK.

[Genetic polymorphism of NFKB1 gene in Chinese Han of Chengdu and Thai populations].

OBJECTIVE: To investigate the single nucleotide polymorphisms (rs3774963 C>G and rs11722146 A>G) of NFKB1 gene between the Chinese Han of Chengdu and Thai populations, and simultaneously to compare distributions of genotype and allelic frequencies of NFKB1 among different ethnic groups from the International Haplotype Map Project. METHODS: The genotypes and allele frequencies of NFKB1 gene rs3774963 C>G and rs11722146 A>G were analyzed in 118 healthy Chinese Han of Chengdu and 101 Thai individuals using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) strategy and DNA sequencing. RESULTS: The frequencies of the CC, CG, and GG genotypes of rs3774963 C>G were 15.3%, 43.2%, and 41.5% in Chinese Han of Chengdu, and 25.7%, 47.5%, and 26.7% in Thai population, respectively. The frequencies of the C and G alleles were 36.9% and 63.1% in Chinese Han of Chengdu, and 49.5% and 50.5% in Thai population, respectively. There were significant differences in the genotypes and allele frequencies between the two groups. The frequencies of the AA, AG, and GG genotypes of rs11722146 A>G were 22.9%, 50.0%, and 27.1% in Chinese Han of Chengdu, and 18.8%, 53.5%, and 27.7% in Thai population, respectively. The frequencies of the A and G alleles were 47.9%, 52.1% in Chinese Hen of Chengdu, and 54.5%, 45.5% in Thai population, respectively. However, no statistically significant difference was observed between the two populations. Interestingly, when compared with the data from the International Haplotype Map Project, the genotypes and allele frequencies of NFKB1 gene rs11722146 A>G but not rs3774963 C>G in the Chinese Han of Chengdu were significantly different from those among European and Sub-Saharan African populations. CONCLUSION: NFKB1 gene polymorphism in diverse populations is significantly different.

[Study on genetic polymorphisms of P-selectins in Chinese Han of Chengdu and Thai populations].

OBJECTIVE: To study the gene polymorphisms of position --2123 C/G,--1969 G/A,--1817 T/C in promoter region and of Thr715Pro in exon thirteenth of P-selectin in the Chinese Han of Chengdu and Thai populations, and simultaneously to compare distributions of genotype and allelic frequencies of P-selectins among different races. Methods The genotypes and allele frequencies of the P-selectin base --2123 C/G,--1969 G/A,-1817 T/C and amino acid Thr715Pro were detected by polymerase chain reaction -restriction fragment length polymorphism (PCR-RFLP) to 120 healthy Chinese Han of Chengdu and 110 Thai population. RESULTS: There were no significant differences in the genotype and allele distribution of--2123 C/G,--1969 G/A,--1817 T/C polymorphisms for the P-selectin gene between Chinese Han of Chengdu and Thai populations (P > 0.05), in which compared with England and American, the distribution of P-selectin genotype and allele had significantly differences among ethnics (P < 0.001). No polymorphism of Thr715Pro was found in this study. Conclusion In Chinese Han of Chengdu and Thai populations the polymorphisms exist at base position--2123 C/G,--1969 G/A and --1817 T/C in promoter region of P-selectin. There are no significant differences in the genotype and allele distribution of the P-selectin gene polymorphisms between Chinese Han of Chengdu and Thai populations, but significantly different distribution of P-selectin gene polymorphisms occur among ethnics.

[Therapeutic effect of a new recombinant immunotoxin mMIP-1alpha-DT390 on experimental autoimmune encephalomyelitis].

OBJECTIVE: To evaluate the therapeutic effect of a new recombinant immunotoxin mMIP-1alpha-DT390 on experimental autoimmune encephalomyelitis (EAE). METHODS: EAE was induced in the low-sensitive strain C57BL/6 mice with intraperitoneal injection of myelin basic protein (MBP) to simulate the human disease multiple sclerosis, followed by intramuscular injection of cationic liposome carrying the plasmid DNA SRalpha-mMIP-1alpha-DT390 in the leg muscle to elicit resistance to EAE development. The mice were then examined daily for clinical signs of EAE by an observer blind to the treatment protocol. For immunohistochemistry the mice were anesthetized and perfused with sterile PBS and paraformaldehyde, and the cerebrum, cerebellum, medulla and spinal cord were removed for preparation of serial sections. The mononuclear cells (MNCs) from the EAE mouse spleens were prepared for three-color flow cytometry analysis of the surface markers with appropriate antibodies following the BD Pharmingen cytokine staining protocol. RESULTS: EAE model was successfully established by active MBP immunization in C57BL/6 mice. Administration of the immunotoxin mMIP-1alpha-DT390 significantly delayed the disease onset and lowered the mean clinical score for EAE as compared with the control mice. Immunohistochemistry demonstrated much less CCR5(+) infiltrating cells in the central nervous system in mMIP-1alpha-DT390-treated mice than in the control. The treatment also eliminated reactive T cells in the periphery blood without affecting the number of B cells. CONCLUSION: The immunotoxin mMIP-1alpha-DT390 can attenuate the disease activity of EAE in mice, suggesting its potential use in the treatment of other autoimmune disorders.

[Induction of hypoxia-inducible factor-1alpha in two kinds of rats asphyxiation death models].

OBJECTIVE: To investigate the expression of hypoxia-inducible factor 1-alpha (HIF1-alpha) in the heart, lung, liver and kidney in rats died of two typical models of asphyxia. METHODS: Two asphyxia models were made and tissue samples of the dead rats were collected from different groups at various postmortem duration. The expression and the changes of HIF1-alpha in various tissues were examined by immunohistochemistry and image analysis techniques. Results Significant expression of HIF1-alpha was observed in the myocardial fibers, kidney cells, liver cells and lung cells in both asphyxia models, but not in the control group. The expression of HIF1-alpha in various tissues in the rat died of nitrogen gas breathing was found in the nuclei at 0 hour and the expression level decreased gradually thereafter. The HIF1-alpha expression level and duration in various tissues of the rat died of hanging were higher and longer than that of the former group, with a peak of the expression level observed 6 hours after death, and then started to decline in all tissues except the heart where the expression still showed an increase 24 hours after death. The control groups showed a steady expression in the cytoplasm but not in the nuclei. CONCLUSION: HIF1-alpha appears to be a valuable biomarker in the diagnosis of asphyxia within 24 hours after death.

[Primary study of the recombinant immunotoxin DT390-mRantes in EAE therapy].

AIM: To construct a novel eukaryotic expression plasmid including the recombinant immunotoxin DT390-mRantes and treat experimental autoimmune encephalomyelitis (EAE) in mice. METHODS: EAE in C57BL/6 mice were induced by the extracted MBP. The mRantes fragment was inserted into the eukaryotic expression plasmid SRalpha containing DT390. Then cationic liposome-embedded plasmid DNA was injected into the muscles of the hind-limbs in mice. The effect of DT390-mRantes was evaluated by observing clinical symptoms, pathological changes of brain, relative cytokine of peripheral blood, and the proportion of T cells and B cells. RESULTS: The recombinant immunotoxin DT390-mRantes was successfully constructed. Compared the mice in treated group with those in untreated group the clinical symptoms of EAE were alleviated, the infiltration of inflammatory cells were decreased, the IFN-gamma level was fallen, and the ratio of T/B cells was decreased. CONCLUSION: The recombinant immunotoxin DT390-mRantes has distinct effects on EAE in mice, which may be used for beneficial reference to the therapy of MS.

[The research on construction and expression of eukaryotic expression plasmid for a recombinant immunotoxin mMIP-1alpha-DT390].

OBJECTIVE: To construct a novel recombinant immunotoxin (IT) expression vector by fusing mouse macrophage inflammatory protein-1alpha gene (mMIP-1alpha) into a truncated diphtheria toxin gene (DT390), and examine the expression of mMIP-1alpha-DT390 fusion protein in NIH3T3 cells. METHODS: mMIP-1alpha cDNA was cloned from mouse liver tissue through reverse transcription-polymerase chain reaction (RT-PCR), and inserted in the expression plasmid SRalpha, that includes the DT390 gene, to form the recombinant vector SRa-mMIP-1alpha-DT390. The positive recombinant plasmid was identified by PCR, the restriction endonucleases digestion and DNA sequencing, and then by liposome protocol, the identified positive plasmid was transferred into NIH3T3 cells for observing the fusion protein expression by immunofluorescence, with detecting the activities of the immunotoxin in vitro through MTT. RESULTS: We have successfully constructed a recombinant immunotoxin expression vector named as SRalpha-mMIP-1alpha-DT390. The immunofluorescence photograph sourced from fluorescence immunocytochemical method showed that the fusion gene could express in the cytomembrane and cytoplasm of NIH3T3 cells. In the bioactivity detection assay, the supernatant of the transfected cell culture was observed to have the obvious cytotoxic activity to activated T-lymphocyte. CONCLUSION: The SRalpha-mMIP-1alpha-DT390 recombinant immunotoxin expression vector will provide the basis of studying the targeted cytotoxic activity to inflammatory cells, and may have some potential value for clinical application.

[Genetic polymorphisms of three loci and implication to forensic medicine].

OBJECTIVE: To obtain population genetic data of short tandem repeat (STR) locus D2S1327, D1S1390, and D11S2008 and to investigate the disparity of allelic frequency distributions among populations from different regions. METHODS: Blood samples of 300 unrelated individuals from Chengdu (Han), Bangkok (Thai) and Maint (Germany) were taken and analyzed with single PCR, polyacrylamide gel electrophoresis and silver staining. RESULTS: In the three loci, 9, 6, and 8 alleles and 32,14, and 22 genotypes were found respectively. The observed heterozygosity of 79%-82%, 63.0%-74.3%, and 72.0%-74.3% and discrimination power of 87.8%-92.6%, 79.6%-82.5%, and 87.6%-89.0% were identified for the three loci respectively. The genotype distributions of the three loci in the three populations fitted well with Hardy-Weinberg equilibrium. There was no significant difference in allelic frequency distributions among the three populations. CONCLUSION: The methods described in this paper are easy to perform and have high sensitivities. The discrimination power and exclusion chances of these three loci are desirable for forensic analysis and application.

[Study of the construction and function of an eukaryotic expression plasmid of immunotoxin DT390 gene].

AIM: To construct a new recombinant eukaryotic expression plasmid of murine IFN-gamma-inducible protein 10 (mIP10) gene and a truncated diphtheria toxin (DT390) gene and investigate its biological function. METHODS: mIP10 cDNA was cloned from murine liver tissue by RT-PCR and was inserted into eukaryotic expression plasmid SRalpha containing DT390 gene to construct plasmid SRalpha-DT390-mIP10. After being identified with restriction endonucleases digestion analysis and DNA sequencing, the recombinant plasmid was transfected into NIH3T3 cells through PolyFect. The expression of recombinant plasmid was detected by immunofluorescence assay and the bioactivity of mIP10-DT390 in cultured supernatant of the transfected NIH3T3 cells was detected by MTT colorimetry. RESULTS: A new recombinant immunotoxin expression plasmid SRalpha-DT390-mIP10 was constructed successfully and was expressed in NIH3T3 cells. The expressed DT390-mIP10 killed the activated T cells effectively. CONCLUSION: A new recombinant eukaryotic expression plasmid SRalpha-DT390-mIP10 has been constructed and expressed successfully, which is useful in further studying the treatment of cancer and autoimmune diseases.