[Identification of critical influencing factors in dropping process of Xuesaitong Dropping Pills].

With the dropping process of Xuesaitong Dropping Pills(XDP) as the study object, critical factors affecting the quality indicators of pill pass rate, average weight of drop pills and roundness were screened out, so as to deepen the understanding of the dropping process. The critical process units, critical quality attributes and potential critical process influencing factors of XDP were determined by risk analysis and prior knowledge, and then the critical influencing factors were screened out by Plackett-Burman design. First, according to the risk assessment, the critical technique of XDP preparation process was dropping, and then the critical quality attributes of dropping process were pill pass rate, average weight of drop pills and roundness. Then, according to fishbone diagram and failure mode and effects analysis(FMEA), potential critical influencing factors were determined as flow rate, matrix ratio, solid-liquid ratio, feed-liquid temperature, top temperature of condensate, bottom temperature of condensate and dropping distance. Finally, among these seven potential factors, the critical influencing factors were determined as material liquid ratio, dropping distance, top temperature of condensate, bottom temperature of condensate. This study revealed the potential of Plackett-Burman design in screening and understanding the influence of selected factors on XDP dropping process, which could provide a reference for studying the dropping process.

Effects of meglumine cyclic adenylate pretreatment on systemic inflammatory response syndrome induced by lipopolysaccharide in rats.

Studies showed that the use of cyclic adenosine monophosphate (cAMP) substitutes or intracellular cAMP activators increased intracellular cAMP level, causing anti-inflammatory effects. This study was to investigate the effects of pretreatment with meglumine cyclic adenylate (MCA), a compound of meglumine and cAMP, on systemic inflammation induced by lipopolysaccharide (LPS) in rats. Eighteen adult male Sprague-Dawley rats were randomly divided into 3 groups (n=6 each): control group (NS group), LPS group (LPS group) and LPS with MCA pretreatment group (MCA group). Systemic inflammation was induced with LPS 10 mg/kg injected via the femoral vein in LPS and MCA groups. In MCA group, MCA 2 mg/kg was injected via the femoral vein 20 min before LPS injection, and the equal volume of normal saline was given in NS and LPS groups at the same time. Three hours after LPS injection, the blood samples were taken from the abdominal aorta for determination of plasma concentrations of TNF-α, IL-1, IL-6, IL-10, cAMP by ELISA and NF-κBp65 expression by Western blotting. The experimental results showed that inflammatory and antiinflammatory indices were increased in LPS group compared to NS group; inflammatory indices were declined and anti-inflammatory indices were increased in MCA group relative to LPS group. Our study suggested that MCA pretreatment may attenuate LPS-induced systemic inflammation.

[Progesterone Promotes Human Bone Marrow Mesenchymal Stem Cells to Synthesize Fibronectin via ERK Pathway].

OBJECTIVE: To investigate whether the progesterone can promote fibronection (FN) synthesis by human bone marrow mesenchymal stem cells (MSCs) and to explore the potential underlying mechanism. METHODS: The human bone marrow MSCs were cultured in a serum-free medium with progesterone for 72 hours, the MTT test was performed to observe the proliferation status and adhension ability of the treated cells. Western blot was used to detect the content of FN in MSDs with GAPDH as the internal reference, the phosphorylation of ERK1/2, as well as the FN content in MSC treated by PD98059, a specific inhibitor of ERK1/2. RESULTS: The progesterone at a range of certain doses not effect on the proliferation of human bone marrow MSCs. Progesterone (25 µg/L) treatment enhanced the FN expression and adherent ability of marrow MSCs. Progesterone could induce prompt phosphorylation of ERK 1/2 and its promoting effects on FN synthesis was reversed by PD98059. CONCLUSION: The progesterone can promote FN synthesis by human bone marrow MSCs via ERK 1/2 pathway, and it might be used to culture MSCs in serum-free medium.

[Putrescine Promotes Human Marrow Mesenchymal Stem Cells to Differentiate along Osteogenic Pathway].

OBJECTIVE: To investigate the effects of putrescine on the growth and differentiation of human bone marrow mesenchymal stem cells (MSC) to develop a new inductive medium mixture for their osteogenic differentiation. METHODS: Human bone marrow MSC were collected from three healthy donors and were used to observe the growth-promoting activity of putrescine with MTT test. Experiments were divided into 3 groups: (1) putrescine group, (2) positive control group (presence of dexamethasone, ascorbate, and glycerol phosphate) and negative group (d-alpha with 5% FCS). The cellular expression level of Runx-2 was detected by PCR assay after the culture was maintained for 1 week. After 2 weeks, the intracellular activity of alkaline phosphatase was revealed by histochemistry staining, the phosphatase activity, and the protein concentration in the cell lysates were also detected. Furthermore, MSC were cultured in the presence of putrescine for 2 weeks and Oil-red O staining was performed to reveal the differentiated adipocytes; the cells induced by the standard agent cocktail were used as the positive control. RESULTS: Putrescine promoted the proliferation of human marrow MSC in a dose-dependent manner. MSC exposed to putrescine at a concentration of 100 µmol/L for 1 week expressed greatly higher level of Runx-2, compared with the negative control. Alkaline phosphatase activity was evidently observed after MSC were maintained in the presence of putrescine for 2 weeks. The phosphatase activity contrasted to the protein content in putrescine-treated MSC was significantly higher than that of the control cells (0.87±0.012 vs 0.52±0.010) (P<0.01), and also greatly higher than that of the positive control (0.83±0.029) (P=0.02). Oil red O staining showed that MSC treated by putrescine did not differentiate into adipoblasts. CONCLUSION: Putrescine can promote the proliferation and osteogenic differentiation of MSC, suggesting the potential application of putrescine as a novel inductive agent for in vitro osteogenesis of MSC.

Dicaine represses apoptosis-linked gene 2-interacting protein X expression to induce airway epithelial barrier dysfunction.

Epithelial barrier dysfunction is associated with a number of inflammatory disorders. However, the pathogenesis of epithelial barrier dysfunction is unclear. This study aims to elucidate the involvement of dicaine in airway epithelial barrier dysfunction. In the present study, an RPMI2650 (Rpc) human airway epithelial cell line was cultured, with or without dicaine, in monolayers using Transwells. In order to assess airway epithelial barrier function, the levels of transepithelial electrical resistance and permeability to ovalbumin (OVA) were measured. Expression of apoptosis-linked gene 2-interacting protein X (Alix) in Rpc cells was assessed using quantitative reverse transcription­polymerase chain reaction and western blotting. The antigenicity of OVA was assessed using a T cell proliferation assay. The results of the present study demonstrated that Alix expression levels were markedly lower in Rpc cells treated with dicaine, compared with those not treated with dicaine. An increase in the level of transcellular permeability to OVA was observed in Rpc monolayers following treatment with dicaine, compared with that in the Rpc monolayer without dicaine treatment. Furthermore, the Rpc monolayer maintained high antigenicity and induced antigen specific T cell proliferation. In conclusion, dicaine causes a decrease in expression levels of Alix, which resulted in compromise of the Rpc cell monolayer epithelial barrier function.

[Slowly-adhered primary bone marrow mesenchymal stem cells may exhibit more potent immuno-regulatory activity].

OBJECTIVE: This study was to investigate the relationship between the adherent ability of freshly isolated MSCs with their inhibitory effect on lymphocyte activation. METHODS: Human bone marrow mononucleated cells were maintained in culture for 48 hours, the attached and the non-attached cells were then cultured separately and the adherent cells were collected and passaged. Cellular surface markers were analyzed with flow cytometry. Alkaline phosphatase activity and the intracellular lipid droplets were measured by histological staining, the in vitro osteogenesis and adipogenesis were identified. One-way mixed lymphocyte reaction was used to evaluate the suppressive activity of the adherent cells on lymphocyte proliferation, the prostaglandin E2 level in supernatant of cultured cells was detected by ELISA. RESULTS: Some cells attached to the plastic after the bone marrow mononucleated cells were allowed to adhere for 48 hours. The slowly-attached cells were fibroblast-like in morphology, homogenously positive for CD44 and CD73 and negative for CD31 and CD45. They could be coaxed into osteoblasts and adipoblasts under the standard inductive conditions. These cells were able to inhibit lymphocyte proliferation in mixed lymphocyte reaction and their effect was more potent than those from the adherent cells appeared within 48 hours. The concentration of prostaglandin E-2 in the supernatants of the slowly-adhered cells was significantly higher than that in the MSCs cultured with the traditional method (90.8 ± 10.37 ng/ml vs 70.2 ± 8.98 ng/ml) (P < 0.01). CONCLUSIONS: The MSCs exist in the marrow mononucleated cells after adherent culture for 48 hours, and the MSCs may exhibit more potently inhibitory activity on lymphocyte activation.

[Exploration of conditions for releasing microvesicle from human bone marrow mesenchymal stem cells].

The release of microvesicles(MV) is one of the critical mechanisms underlying the angiogenesis-promoting activity of mesenchymal stem cells(MSC). This study was aimed to explore the appropriate condition under which MSC releases MV. Bone marrow samples from 5 healthy adults were collected, and MSC were isolated, culture-expanded and identified. MSC at passage 5 were suspended in medium without or medium with 10% fetal(FCS) calf serum and seeded into culture dishes. The culture was separately maintained in hypoxia (1% oxygen) or normoxia (around 20% oxygen), and 20 dishes of cells (2×10(6)/dish) were used for each group. The supernatants were collected for MV harvesting. The cell number was counted with trypan blue exclusion test and the protein contents in the MV were determined. MV were identified by observation under an electron microscope. The surface markers on MV were analyzed by flow cytometry. MTT test was performed to observe the pro-proliferative activity of MV that were added into the culture of human umbilical cord vein endothelial cells at a concentration of 10 µg/ml. The results showed that the majority of MV released by MSC were with diameters of less than 100 nm, and MV took the featured membrane-like structure with a hypodense center. They expressed CD29, CD44, CD73 and CD105, while they were negative for CD31 and CD45. The increase multiples of the adherent trypan blue-resistant cells cultured in normoxia with serum, in normoxia without serum, in hypoxia with serum and hypoxia in the absence of serum were 4.05 ± 0.73, 1.77 ± 0.48, 5.80 ± 0.65 and 3.69 ± 0.85 respectively, and the estimated protein contents per 10(8) cells were 463.48 ± 138.74 µg, 1604.07 ± 445.28 µg, 2389.64 ± 476.75 µg and 3141.18 ± 353.01 µg. MTT test showed that MV collected from MSC in hypoxia seemed to promote the growth of endothelial cells more efficiently than those from cells in normoxia. It is concluded that hypoxia can enhance the release of microvesicles from MSC, and cultivation of MSC in hypoxia and medium without serum may provide an appropriate condition for MV harvesting.

[Investigation on the relationship between the solar term of onset and syndrome types in 430 patients with acute myocardial infarction by circular statistical analysis].

OBJECTIVE: To study the relationship between the solar term of onset of acute myocardial infarction (AMI) and its syndrome types in traditional Chinese medicine (TCM). METHODS: The clinical data about 430 patients with AMI hospitalized in Foshan Hospital of TCM from February 4th 2003 (Beginning of Spring) to February 3rd 2008 (Beginning of Spring) were collected, and the solar term of onset as angle coordinate was regarded, then the peak phase of the onset solar term in each syndrome type of AMI was calculated by circular statistical analysis. RESULTS: Among 430 patients with AMI, 134 patients were considered to have qi stagnancy and blood stasis syndrome, 188 patients showed the syndrome of turbid sputum obstruction, 29 of them showed deficiency of yin-blood, and 79 showed deficiency of yang qi. The clinical manifestation of AMI was mainly asthenia syndrome (qi stagnancy and blood stasis+turbid sputum obstruction, 74.9%). According to the circular statistical analysis, the peak of the solar terms of AMI onset occurred at the Beginning of Spring in all cases (r=0.127 4, P<0.01), and standard deviation (s)=116.300 6 degree angle, showed it mainly occurred in winter and spring. As the peak of the onset of qi stagnancy and blood stasis occurred at Winter Solstice and Lesser Cold (r=0.200 5, P<0.01), its peak occurred in winter; the turbid sputum obstruction syndrome occurred at Spring Equinox (r=0.147 0, P<0.05), mainly in spring, yet the symptoms of above two peaks were generally mild. Besides, there was no significant difference in onset of the solar term in regard to onset of deficiency of yin-blood and deficiency of yang qi (both P>0.05). CONCLUSION: There is a close relationship between periodicity of the solar terms and onset of AMI. The main treatment for AMI is to expel turbid sputum, activate blood to resolve stasis and promote blood circulation to relieve pain; also the method of activating blood to resolve stasis is frequently contemplated in winter, and the method of expelling turbid sputum is the main strategy in spring.

[Protective effects of nicotine on inflammatory cytokines in myocardial ischemia/reperfusion injury in rats].

OBJECTIVE: To investigate the effect of nicotine on inflammatory cytokines in myocardial ischemia/reperfusion (I/R) injury in rat. METHODS: Fifty male Sprague-Dawley (SD) rats were divided into five groups by random numbers table (each n=10): sham operation group (S group), I/R group, nicotine 400 µg/kg group (H group), nicotine 40 µg/kg group (L group) and α-bungarotoxin (α-BGT,1 µg/kg) group. The anterior descending branch of left coronary artery was occluded for 30 minutes followed by 90 minutes reperfusion to reproduce myocardial I/R injury rat model, while in S group the anterior descending branch of left coronary artery was only exposed without occlusion procedure. Thirty minutes before myocardial ischemia, drugs in corresponding doses were given intravenously via jugular vein. At the end of 90 minutes of reperfusion, blood samples were collected from carotid artery to determine the levels of tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8), IL-10, MB isoenzyme of creatine kinase (CK-MB), and cardiac troponin I (cTnI), then the animals were sacrificed and the hearts were harvested for pathological study and determination of myeloperoxidase (MPO) activity. Immunohistochemistry and reverse transcription- polymerase chain reaction (RT-PCR) were used to assess intercellular adhesion molecule-1 (ICAM-1) protein and mRNA expression in heart tissue. RESULTS: Compared with the S group, the concentrations of TNF-α, IL-8, IL-10, CK-MB, cTnI, MPO activity, ICAM-1 protein and mRNA expression were significantly increased in I/R group [TNF-α (ng/L): 158.7±32.7 vs. 31.5±5.8, IL-8 (ng/L): 0.71±0.06 vs. 0.30±0.04, IL-10 (ng/L): 69.0±7.8 vs. 41.4±4.3, CK-MB (U/L): 2 540±169 vs. 1 120±120, cTnI (µg/L): 26.2±4.6 vs. 0.9±0.2, MPO (U/g): 4.2±0.6 vs. 1.6±0.4, ICAM-1 protein: 0.210±0.025 vs. 0.100±0.018, ICAM-1 mRNA: 1.82±0.23 vs. 1.18±0.20, P<0.05 or P<0.01]. Injury to myocardial ultrastructure was worse in I/R group. Compared with the I/R group, the plasma levels of TNF-α and IL-8 were lower [TNF-α (67.3±9.8) ng/L, IL-8 (0.47±0.04) ng/L], IL-10 was higher [(147.5±12.5) ng/L], CK-MB, cTnI, MPO, ICAM-1 protein and mRNA were lower obviously in H group [CK-MB (1 282±145) U/L, cTnI (4.7±1.4) µg/L, MPO (2.5±0.4) U/g, ICAM-1 protein 0.140±0.026, ICAM-1 mRNA 1.31±0.25, P<0.05 or P<0.01]. Injury to the myocardial ultrastructure was less marked in H group. The indexes of those in L group and α-BGT group compared with I/R group were not statistically significantly different. CONCLUSION: Nicotine can block endothelial expression of adhesion molecules and neutrophil adhesion and infiltration to promote a balance of anti-inflammatory and pro-inflammatory response, thus prevents excessive inflammatory response to myocardial I/R injury in rat.