Effect and Mechanism of Superantigen Staphylococcal Enterotoxin Therapy for Mouse Gastric Tumor

Summary:The anti-tumor effect and mechanism of the staphylococcal enterotoxin A(SEA)werestudied.The mouse gastric tumor model was produced by subcutaneously inoculating gastric tumorcells(MGC80-3).The experimental group was treated with SEA,and the control group was treatedwith normal saline.The percentage of tumor generation and tumor mass was measured.The resultsshowed that the percentage of the tumor tumor generation in the SEA-treated mice was lower than in thecontrol group,but there was no significant difference(P>0.05).However,the tumor mass in theexperimental group was significantly lighter than in the control group,with the difference bing verysignificant(P<0.001).There were more CD_4~+ T cells and CD_8~+ T cells in the tumor of the micetreated with SEA than those of the control group.SEA has an obvious anti-tumor effect on mice gas-tric tumor.The mechanism might be that SEA induces the effect of superantigen-dependent cell me-diated cytotoxicity to the tumor cells.     

Effect of Phosphorus—32 Glass Microspheres on the Human Hepatocellular Carcinoma in Nude Micer

Objective To study anticancer effect and ultrastructural influence of phosphorus-32 glass microspheres(^32P-GMS) injected in the hepatocellular carcinoma in nude mice.Methods The ultrastructural changes of tumor in both the treatment group and control group were examined by transmission electron microscope.Results In the treatment group,a large number of tumor cells were killed and the death rate of tumor cells was much higher(35%-70%).Ultrastructurally,severe nuclear damage was observed in the dead cells.The early characteristics of necrosis such as margination of heterochromatin were also found in some tumor cells.Besides,well-differentiated tumor cells,degenerative tumor cells and some lymphocytes were seen.The skin and muscle close to the tumor were normal.In the control group,the tumor consisted of poor differentiated tumor cells,in which there were only a few appototic cells(5%).Conclusion The results suggest that the local administration of^32P-GMS produces obviously the anticancer effect.     

Comparative Study of the Effect of Shugan Shuru Granule(疏肝舒乳颗粒)on Pathology and p53 Gene Expression in Patients with Hyperplastic Disease of Breast

Objective: To study the effect of Shugan Shuru Granule (疏肝舒乳颗粒, SSG) on the p53 gene expression in patients with hyperplastic disease of breast (HDB) to indirectly explore the mechanism of SSG's effect on HDB on the molecular pathological level. Methods: Sixty-six patients with HDB were allocated in the treated group and the control group, with the former treated with SSG and the latter not. All patients underwent breast operation and their diseased mammary tissues were cut out, sectioned, and observed under microscopy with HE staining and immunohistochemical staining, with ABC method adopted to estimate the degree of hyperplasia and p53 gene expression. The severity of HDB was classified into normal, mild, moderate and severe grades (marked as 0 to Ⅲ ), according to the degree of hyperplasia in the mammary gland.Results: Hyperplasia in the control group mostly belonged to grade Ⅰ - Ⅲ before treatment, showing overgrowth of gland and proliferation of glandular epithelial cells, which were high columnar shaped, more stratified, with papillary or substantive dysplasia. While in the treated group, most belonged to grade 0- Ⅰ after SSG treatment, with proliferated gland and dysplasia recovered to normal or disappeared. The positive rate of p53 gene expression in the treated group was 9.09%, and in the control group 39.39%, comparison between the two groups showing significant difference ( P＜0.01), the intensity in the former was significantly weaker than that in the latter. Conclusion: SSG could not only inhibit the proliferation of mammary duct epithelia and Iobuli, but also inhibit the over-expression of P53. Therefore, it could be regarded as an effective remedy for treatment of HDB and prevention of mammary cancer genesis.     

Effects of lptakalim on intracellular free calcium concentration of cultured rabbit pulmonary arterial smooth muscle cells

Objective：To explore the effects of Iptakalim on intracellular free calcium concentration and on the proliferation of cultured rabbit pulmonary arterial smooth muscle cells induced by endothelin-1 （ET-1） in vitro. Methods：A cell culture model, [^3H]-thymidine（[^3H]-TdR） incorporation test and confocal microscope were used to observe proliferation and intracellular free calcium concentration（[Ca^2±]） of rabbit PASMC induced by ET-1 in vitro. Results：The value of [^3H]-TdR incorporation in ET-1 group was increased 1.468 times higher than that in control group. Iptakalim at the concentration of 10^-7mol/L, 10^-6mol/L ,10^-5 mol/L lowered [^3H]-TdR incorporation by （19.8 ± 4.6）%, （41.2 ± 9.5）%, （54.7 ± 10.1）%, respectively, compared with the value of the cells treated with ET-1（P〈 0.01）; The intracellular fluorescence intensity of PASMC in ET-1 group was increased from 73.70 ± 10.12 to 143.84 ± 28.23, significantly higher than that in control group（P 〈 0.01）; whereas with Iptakalim,the fluorescence intensity（FI） was only increased from 74.30 ± 10.20 to 86.03 ± 9.82, significantly lower than that in ET-1 group（P 〈 0.01）. Conclusion：Iptakalim inhibited proliferation of PASMC and decreased intracellular free calcium concentration of cultured rabbit PASMC induced by ET-1.     

Inhibitory Effect of Melatonin on the Growth of H22 Hepatocarcinoma Cells by Inducing Apoptosis

Whether melatonin not only inhibits the growth of H22 hepatocarcinoma cells but also induces apoptosis in vitro was assessed. The anti-proliferative effects of melatonin on tumor cells was observed by MTT assay and tumor cells growth curve assay. And the apoptosis of the cells was studied by acridine orange fluorescence assay and flow eytometry. The cell cycle of the tumor cells was also observed by flow eytometry. It was found that melatonin could significantly inhibit the growth of H22 hepatoeareinoma cells. Incubated with melatonin, ehromatin condensation of the tumor cells was observed by fluorescence microscopy. Compared with control, the percentage of apoptotic cells was increased, and the proportion of G0/S increased but that of G0/M decreased. It was suggested that melatonin could directly inhibit the growth of H22 hepatoearcinoma cells by inducing apoptosis and extending the length of cell cycle of the tumor cells.     

Clinical study on effect of Astragalus Injection (黄芪注射液) and its immuno-regulation action in treating chronic aplastic anemia

Objective:To observe the clinical effect of Astragalus Injection(黄芪注射液,AI) and its immuno-regulatory action in treating chronic aplastic anemia(CAA).Methods:Sixty patients with CAA were randomly assigned to two groups equally,both were treated with Stanozolol three times a day,2 mg each time through oral intake,but AI was given additionally to the patients in the treated group once a day via intravenous dripping.All were treated for 15 days as one therapeutic course and the whole medication lasted for more than 4 months totally,with follow-up adopted.The clinical effi cacy was estimated and the changes of T-lymphocyte subsets in peripheral blood as well as the serum levels of tumor necrosis factor-α(TNF-α) and interleukin-2(IL-2) were observed.Results:The total effective rate in the treated group was 83.3%(25/30),which was higher than that in the control group 66.7%(20/30),showing significant difference between them(P<0.05).Levels of hemoglobin,WBC,reticular cell and platelet were elevated in both groups after treatment,but the improvement was signif icantly better in the treated group than that in the control group with respect to the former three indexes(P<0.05).The level of CD4 increased and that of CD8 decreased signifi cantly after treatment in the treated group(P<0.05),which showed significant difference as compared with those in the control group(P<0.05).Levels of serum TNF-α and IL-2 lowered after treatment in both groups,but signifi cance only showed in the treated group(P<0.05).The degree of proliferation in bone marrow got raised signifi cantly and the percentage of non-hemopoietic cells reduced signifi cantly in the treated group after treatment,also showing significant difference to those in the control group(P<0.05).Conclusion:AI could promote the recovery of hemopoietic function,which might be through improving T-lymphocyte subsets and reducing the release of negative regulatory factors such as TNF-α and IL-2 to alleviate the inhibition on hemopoietic function.     

The cellular and molecular mechanism of laminin-glycopeptides on anti-metastasis

Objective To further study the anti-metastasis mechanism of laminin-glycopeptides on carcinoma cell proliferation, apoptosis and the secretion of matrix metalloproteinases. Methods Human hepatocellular carcinoma cells in serum free medium were incubated on laminin-coated substrate with or without laminin-glycopeptides at a final concentration of 50 μg/ml. The total number of surviving cells after incubating for the indicated time was assayed by MTT assay. DNA synthesis of the incubated cells was detected by (3)H-TdR incorporation.Cell cycle was analysed by FACS.The mitotic index of Giemsa stained cells was assessed.Cell apoptosis was detected by both FACS and an acridine orange staining method.Matrix metalloproteinase secretion was analysed by gelatin zymography. Results The total number of surviving cells incubated on laminin in the absence of laminin-glycopeptides was significantly larger than that in the presence of laminin-glycopeptides. Laminin promoted (3)H-TdR incorporation of carcinoma cells, decreased the percentage of cells in G1 phase and increased the percentage of cells in S phase.In contrast, laminin-glycopeptides could inhibit the effect of laminin as shown by (3)H-TdR incorporation and cell cycle analysis. The percentage of cells in G2+M phase and the mitotic index among various groups showed no significant difference.Matrix metalloproteinases secretion from cells treated by laminin-glycopeptides was much less compared to that without the treatment by laminin-glycopeptides. Conclusion Laminin may stimulate cell proliferation, while laminin-glycopeptides could significantly inhibit the effect of laminin by inhibiting DNA synthesis and arresting the carcinoma cell cycle from G1 to S phase.These effects may inhibit not only tumor growth of the primary carcinoma, but also the establishment of metastases at ectopic tissues. Laminin-glycopeptides could also inhibit the secretion of matrix metalloproteinases from carcinoma cells and this may contribute to their decreased invasive and metastatic phenotype. This study further revealed the cellular and molecular mechanism of laminin-glycopeptides on anti-metastasis.     

Effects of Iptakalim on intracellular free calcium concentration of cultured rabbit pulmonary arterial smooth muscle cells

Objective:To explore the effects of Iptakalim on intracellular free calcium concentration and on the proliferation of cultured rabbit pulmonary arterial smooth muscle cells induced by endothelin-1(ET-1) in vitro. Methods:A cell culture model, [3H]-thymidine([3H]-TdR) incorporation test and confocal microscope were used to observe proliferation and intracellular free calcium concentration([Ca2 ]i) of rabbit PASMC induced by ET-1 in vitro. Results:The value of [3H]-TdR incorporation in ET-1 group was increased 1.468 times higher than that in control group. Iptakalim at the concentration of 10-7mol/L,10-6 mol/L,10-5 mol/L lowered [3H]-TdR incorporation by (19.8±4.6)%, (41.2±9.5)%, (54.7±10.1)%, respectively, compared with the value of the cells treated with ET-1(P＜0.01); The intracellular fluorescence intensity of PASMC in ET-1 group was increased from 73.70±10.12 to 143.84±28.23, significantly higher than that in control group(P＜0.01); whereas with Iptakalim,the fluorescence intensity(FI) was only increased from 74.30±10.20 to 86.03±9.82, significantly lower than that in ET-1 group(P＜0.01). Conclusion:Iptakalim inhibited proliferation of PASMC and decreased intracellular free calcium concentration of cultured rabbit PASMC induced by ET-1.     

The Observation of the Photodynamic Effect of Hematoporphyrin Derivative (HpD) Combined with Aidiron on Ascitic Hepatoma Cells in Vitro

In this study, we found, through biochemical synthesis of DNA with the incorporation of~3HTdR and morphological observation of the tumor cells, that aidiron (912), an extract fromearthworm, had a stronger kilting effect (27%) on mouse ascitic hepatoma tumor cells (H_(22)) invitro, and that laser-hematoporphyrin derivative (HpD) combined with 912 markedly enhancedthe kilting effect (74%) on tumor cells. By means of the fluoresoence spectrophotography we also dis-covered that the killing effect of laser-HpD-912 on tumor calls was closely related with the produc-tion of free radicals and lipid peroxidation.     

The Effect of Ginkgo Biloba Extract on the Expression of PKCα in the Inflammatory Cells and the Level of IL-5 in Induced Sputum of Asthmatic Patients

To investigate the effect of the Ginkgo Biloba Extract （GBE） on the asthma and examine its possible mechanisms, 75 asthma patients were divided into 4 groups and the patients were respectively treated with fluticasone propionate for 2 weeks or 4 weeks, or treated with fluticasone propionate plus GBE for 2 weeks or 4 weeks. Fifteen healthy volunteers served as healthy controls. Sputum inhalation with inhaling hypertonic saline （4%-5%） was performed. Lung ventilatory function and forced expiratory volume in one second （FEVI） were measured. The numbers of different cells in induced sputum were calculated. The expression of PKCα in the cells was immunocytochemically detected and the percentages of positive cells in different cells were counted. Interleukin-5 （IL-5） in sputum supernatants was detected with enzyme-linked immunosorbent assay. The percentage of eosinophils, lymphocytes, PKCα positive inflammatory cells and the concentration of IL-5 in asthmatic patients were higher than those in the controls （P〈0.05）, and the eosinophils, lymphocytes, positive expression of PKCα and the level of IL-5 were significantly decreased in asthmatic patients after they were treated with fluticasone propionate or fluticasone propionate plus GBE. However, they were still significantly higher than those of the controls. Compared to the group treated with glucocorticosteroid for 2 weeks, no significant decrease was found in the percentage of eosinophils, lymphocytes, PKCα positive inflammatory cells and the IL-5 in the supernatant of induced sputum. Compared with the group treated with glucocorticosteroid for 2 or 4 weeks, significant decrease in the same parameters was observed in the group treated with fluticasone propionate and GBE for 4 weeks. The IL-5 level in the supernatant of induced sputum was positively correlated with the percentage of PKCα-positive inflammatory cells and the percentage of eosinophils in the induced sputum in asthma patient groups respectively （n=150, r=0.83, P〈0.01; n=150,     

Neurotoxic effect of rotenone on dopaminergic neuron PC12 in vitro

Objective： To investigate the mechanism of oxidative stress in rotenone neurotoxicity to dopaminergic neuron PC12. Methods： High differentiated PC12 cells as dopaminergic neurons were treated by different concentrations of rotenone. The morphology was observed with inverted phase contrast microscope and transmission electron microscope. Cell viability and proliferation inhibition were assessed by MTT. SOD and MDA were detected with biochemical assay. And the specific fluorescent probe （DCF-DA） was used to examine ROS in PC12 cells. Results： After treated with rotenone for 24 h, most of the PC12 cells became smaller and rounder. The process of axon was reduced, shortened or broken in a time and concentration dependent manner. The mitochondrial structure and metabolism were changed. Endoplasmic reticulum expanded and the free ribosome increased. Compared with the control group, cell proliferation inhibition increased and cell viability decreased. SOD increased and MDA decreased. The intensity of fluorescence was more obvious in PC12 cells treated by rotenone compared with control group. Conclusion： Rotenone is neurotoxic to cultured dopaminergic neuron PC12. Rotenone might exert this effect through the metabolism of oxidative stress on the pathogenesis of the neuron.     

Clinical Study of Qingluo Tongbi Granules in Treating 63 Patients with Rheumatoid Arthritis of the Type of Yin-deficiency and Heat in Collaterals

The study is to observe the therapeutic effects of Qingluo Tongbi Granules (清络通痹颗粒QTG) in patients with rheumatoid arthritis (RA) and the changes of immune indexes. In this series there are 63 patients with RA of the type of yin-deficiency and heat in collaterals treated with QTG as the treated group and 55 patients of the same type treated with Tripterygium glycosides as the control group. As a results, in the treated group, the curative rate is 9.52% and markedly effective rate 38.10%, with a total effective rate of 90.48%, while the corresponding rates in the control group are 0, 20.00% and 83.64%, respectively. The curative effect in the treated group is better than that in the control group (P<0.05). Besides, no obvious adverse reactions are found in the treated group. Therefore it is concluded that as a new medicinal preparation QTG is safe and effective in the treatment of RA.     

Expression of Beclin1 in osteosarcoma and the effects of down-regulation of autophagy on the chemotherapeutic sensitivity

To explore the expression of Beclin1 in osteosarcoma and investigate the effects of down-regulation of autophagy on the chemotherapeutic sensitivity to cisplatin （DDP）, the expression of Beclin1 in 28 specimens of osteosarcoma （group A） and 19 specimens of normal bone tissues （group B） were immunohistochemically detected. The expression of Beclin1 mRNA in MG63 cells treated with different concentrations of DDP was examined with RT-PCR. After down-regulation of autophagy in MG63 cells by an autophagy inhibitor, 3-methyladenine （3-MA）, the cell proliferation inhibition rate of MG63 cells treated with DDP was evaluated by using the MTT assay. The positive rates of Beclin1 were 67.85% in group A and 94.73% in group B. Its expression was lower in osteosarcoma than in normal bone tissues, with a significant difference found between them （P〈0.05）. RT-PCR showed that the expression of Beclin1 mRNA in the cells treated with high-dose DDP were higher than that in the non-treated cells, and no significant difference in the expression of Beclin1 mRNA was found between the cells treated with low-dose DDP and the non-treated cells. There was a positive correlation between the level of Beclin1 mRNA expression and the concentration of DDP. MTT assay showed that the proliferation inhibition rates of the cell treated with 3-MA and DDP combined were substantially increased when compared with those treated with DDP alone （P〈0.01）. This study demonstrated that autophagy may be implicated in the carcinogenesis of osteosarcoma, and DDP may induce autophagy in the MG63 cells. It also suggests that the down-regulated autophagy could increase chemotherapeutic sensitivity of DDP to osteosarcoma.     

Synthetic Smac Peptide Enhances Chemo-sensitivity of Bladder Cancer Cells

The effects of synthetic Smac peptide （SmacN7） on chemotherapeutic sensitivity of bladder cancer cells were investigated. SmacN7 penetratin peptide was synthesized and delivered into T24 cells. MTT assay was used to evaluate the viability of T24 cells induced by low-dosage of MMC Flow cytometry was used to analyze the proportions of apoptosis. Western blot was used to detect the expression of XIAP and Caspase-3. The activity of Caspase-3 was measured and the effect of SmacN7 combined with MMC on T24 cell lines was also determined. The results showed that SmacN7 penetratin peptide could successfully interact with endogenous XIAP, increase the proportions of apoptosis of T24 cell lines induced by low-dosage of MMC in a dose-and time-dependent manner. An obvious down-regulation of XIAP expression and up-regulation of Caspase-3 was identi-fied by Western blot. The activity of Caspase-3 in experimental group was significantly increased as compared with that in the control group. As compared with MMC group, the viability of T24 cells in SmacN7 penetratin peptide＋MMC group was markedly decreased to 2.22 and 3.61 folds at 24 h and 48 h respectively. It was concluded that SmacN7 penetratin peptide could act as a cell-permeable IAP inhibitor, inhibit the proliferation, induce apoptosis and enhance the chemo-sensitivity of bladder cancer cells to MMC. These findings indicate that SmacN7 penetratin peptide may be a very promising ageut for bladder cancer treatment when used in combination with chemotherapy.     

The Expression and Activity of MMPs Are Increased in Residual Tumor Tissues after the Termination of Immunotherapy

To investigate the invasive ability of the residual tumor cells after immunotherapy and explore the feasible approach suppressing the invasion, mice were inoculated with B16 cells, and then treated by gene therapy with p4-1BBL/psPD-1 or IFN-γ. The production and activities of MMP-9 and MMP-2 in residual tumor tissues were analyzed with gelatin zymography 1 day and 7 days after the termination of the immunotherapy. The production of MMP-9 and MMP-2 by B16 cells treated with IFN-γ was also analyzed. IFN-γ-treated B16 cells were inoculated to mice via subcutaneous injection. The invasion of tumor to muscular tissue was analyzed. Gene therapy with CH50 was used to suppress the invasive growth of tumor. The results showed that the expression and the activities of MMP-9 and MMP-2 were significantly increased 7 days after the end of immunotherapy. The re- sponse of tumor cells to ECM molecules was intensified after the removal of IFN-γ, resulting in significant increase of both the production and activities of MMP-9 and MMP-2, and the increased invasion of tumor. Gene therapy with CH50 effectively suppressed the invasive growth of tumor. It is concluded that the termination of immunotherapy may result in a higher metastatic potential of residual tumor cells. Suppressing tumor invasion by suitable treatment will improve the efficacy of immunotherapy..     

Effect of Quercetin on Proliferation and Apoptosis of Human Nasopharyngeal Carcinoma HEN1 Cells

The effect of quercetin （Que） on proliferation and apoptosis of human nasopharyngeal carcinoma HEN1 cells was investigated. Inhibition rate of quercetin on HEN1 was assayed by MTT method, apoptosis by flow cytometry （FCM）, and the caspase-3 expression of each group by colorimetry set respectively. Quercetin inhibited HEN1 cells in in a dose-（r=0.709, P〈0.01） and time-dependent manner （r=0.703, P〈0.01）. The ratio of apoptotic and necrosis cells was increased in the cells treated with quercetin. Cell cycle was specificly arrested in G2/M phase. Apoptosis cusp was revealed by FCM. The activity of caspase-3 was significantly up-regulated in 5 groups treated with quecetin as compared with control group （P〈0.05）. It was concluded that the growth inhibition of quercetin was highly related to cell cycle arrest at the G2/M phase and induction of caspase-dependent apoptosis in human nasopharyngeal carcinoma HEN 1 cells.     

Effect of Xin’ anning Nasal Drop (心安宁滴鼻剂) in treating coronary heart disease with unstable angina pectoris

Objective： To observe the clinical efficacy of Xin＇anning Nasal Drop （XAND,心安宁滴鼻剂） in treating coronary heart disease with unstable angina pectoris （CHD-UAP）. Methods： Sixty patients with CHD-UAP were assigned, according to the randomizing number table, to two groups, the control group treated with conventional Western medicine, and the treated group treated with conventional Western medicine plus XAND. The clinical efficacy and the changes of S-T segment in resting EKG and total ischemia burden （TIB） in 24-h dynamic EKG were observed. Results： The clinical efficacy, including the effect of angina alleviation, its initiation, and the effect of TOM syndrome score reduction, were significantly superior in the treated group to those in the control group （ P〈0.05 or P〈0.01 ）. The degree and extent of myocardial ischemia were significantly improved in both groups （ P〈0.01 ）, but the improvement in the treated group was better than that in the control group （ P〈0.05）. Moreover, it was worth mentioning that the immediate effect in the treated group was better than that in the control group, and the reduction of TIB, the improvement in heart rate and myocardial oxygen consumption （ immediately after the first administration or by the end of the therapeutic course）, and systolic blood pressure after treatment in the former were all superior to those in the latter, showing significant difference （ P〈0.05 or P〈0.01 ）. Conclusion： XAND has a quick effect in alleviating angina in patients with CHD-UAP, and it is worthy of further studies and spreading in clinical practice.     

Effect of Xin''''anning Nasal Drop ( 心安宁滴鼻剂 ) in Treating Coronary Heart Disease with Unstable Angina Pectoris

Objective: To observe the clinical efficacy of Xin'anning Nasal Drop (XAND, 心安宁滴鼻剂 )in treating coronary heart disease with unstable angina pectoris (CHD-UAP). Methods: Sixty patients with CHD-UAP were assigned, according to the randomizing number table, to two groups, the control group treated with conventional Western medicine, and the treated group treated with conventional Western medicine plus XAND. The clinical efficacy and the changes of S-T segment in resting EKG and total ischemia burden (TIB) in 24-h dynamic EKG were observed. Results: The clinical efficacy, including the effect of angina alleviation, its initiation, and the effect of TCM syndrome score reduction, were significantly superior in the treated group to those in the control group ( P＜0.05 or P＜0.01 ). The degree and extent of myocardial ischemia were significantly improved in both groups ( P＜0.01 ), but the improvement in the treated group was better than that in the control group ( P＜0.05). Moreover, it was worth mentioning that the immediate effect in the treated group was better than that in the control group, and the reduction of TIB, the improvement in heart rate and myocardial oxygen consumption (immediately after the first administration or by the end of the therapeutic course), and systolic blood pressure after treatment in the former were all superior to those in the latter, showing significant difference ( P＜0.05 or P＜0. 01 ). Conclusion: XAND has a quick effect in alleviating angina in patients with CHD-UAP, and it is worthy of further studies and spreading in clinical practice.