| Objectives:To observe the effect of the preprocessing of medicated serum in Xintongshu on NF-κBp65, the post-activation regulated Inflammatory cytokines (TNF-αand IL-1β) of the newborn rat's myocardial cell of hypoxia and reoxygenation; to probe into the machanism of Xintongshu's protective effect on the newborn rat's myocardial cell of hypoxia and reoxygenation by inhibiting its inflammatory reaction channel.Methods:①The primary culture and identification of myocardial cells of the newborn rat:SD rat which was born within 12 to 24 hours was selected and its myocardial cells were cultivated for 48 hours and then the pulsation of myocardial cells was observed. The identification of cTnT was detected by using immunohistochemical method.②Medicated serum in Xintongshu was processed and the inhibiting rate of medicated serum in Xintongshu with different concentrations on the myocardial cell was detected with MTT. The toxicity of myocardial cells was investigated and an optimal concentration which is nontoxic to the myocardial cell was selected based on the results and determinated as the concentration of the next step in the experiment.③The cultivated 72-hour newborn rats'myocardial cells were selected and randomly divided into four groups----the normal control group,H/R group,GSTT control group and the preconditioning group of medicated serum in Xintongshu(Xintongshu group).10% serum of rat was added to the complete cultivation media in the normal control group and was undergone a continuous routine culture for 24 hours. H/R group: myocardial cells were in hypoxia for 1 hour and in reoxygenation for 2 hours. GSTT control group:After GSTT(50mg/L) was added to the cultivation liquid, myocardial cells were in hypoxia for 1 hour and in reoxygenation for 2 hours. Xintongshu group:After 10% medicated serum in Xintongshu was added to the liquid medium, myocardial cells were in hypoxia for 1 hour and in reoxygenation for 2 hours. The shape of the myocardial cells was observed with Rhodamine B Dyeing, the content of MDA and the vitality of SOD were determined with Colorimetry, the content of TNF-a and IL-1βwas determined with ELISA and the expression level of NF-κBp65 mRNA was detected with RT-PCR.Results:1.After 48-hour cultivation, myocardial cells were interwovon into a net, and gradually into cell clusters through pseudopod cells with a synchronizing, regular and vigorous pulsation and a adherent growth. No cells were found dead. The result of identification with immunohistochemical method was posivitve and the purity of myocardial cells reached 95%.2. No statistical differences were observed bewteen the absorbance value of medicated serum with a concetration less than 10% and that of cell control group (P>0.05), statistical differences were observed between the absorbance value of the physic liquor with a concentration more than 15% and that of cell control group (P<0.05). The inhibiting rate of medicated serum with 10% concentration was-1.08%. The results showed that the 10% concentration was the optimal one which was non-toxic to myocardial cells.3. a. Cell shape:Compared with the normal control group, refraction of cell body decreased, ecphyma shortened or disappeared, pulsation weakened or disappeared inH/R group. Compared with H/R group, the shape of myocardial cell in GSTT control group and Xingtongshu group obviously improved. Compared with GSTT control group, the shape of myocardial cell obviously improved. b. Determining the content of MDA and the vitality of SOD with Colorimetry:Compared with the normal control group, the content of MDA obviously increased and the vitality of SOD obviously decreased in H/R group,Statistical differences were observed (P<0.01).The content of MDA decreased and the vitality of SOD increased in GSTT control group and Xingtongshu group,No statistical differences were observed(P>0.05). Compared with H/R group, the content of MDA decreased and the vitality of SOD increased in GSTT control group and Xingtongshu group,statistical differences were observed, statistical differences were observed (P<0.01).Compared with GSTT control group, the content of MDA obviously decreased and the vitality of SOD obviously increased in Xingtongshu group,statistical differences were observed(P<0.01).c. Determining the content of TNF-a and IL-1βwith ELISA:Compared with the normal control group, the content of TNF-a and IL-1βin H/R group obviuosly increased, Statistical differences were observed (P<0.01),the content of TNF-a and IL-1βin GSTT control group and Xingtongshu group decreased,no statistical differences were observed (P>0.05). Compared with H/R group, TNF-a and IL-1βin GSTT control group and Xingtongshu group obviously decreased, statistical differences were observed (P<0.01).Compared with GSTT control group, the content of TNF-a and IL-1βdecreased in Xingtongshu group, no statistical differences were observed (P>0.05). d. Detecting the expression level of NF-κBp65 mRNA with RT-PCR: Compared with the normal control group, the expression level of NF-κBp65 mRNA in H/R groupincreased,statistical differences were observed (P<0.01).The expression level of NF-KBp65 mRNA in GSTT control group and Xingtongshu group decreased, no statistical differences were observed (P>0.05).Compared with H/R group, the expression level of NF-κBp65 mRNA in GSTT control group and Xingtongshu group obviously decreased Statistical differences were observed, statistical differences were observed(P<0.01).Compared with GSTT control group, the expression level of NF-KBp65 mRNA in Xingtongshu group decreased,statistical differences were observed (P<0.05). Conclusion:The preconditioning of medicated serum in Xintongshu can exert a protective effect by inhibiting the inflammatory reaction channel of the myocardial cell of hypoxia and reoxygenation. Its mechanism may be related to the decrease of the expression level of NF-KBp65 mRNA and the content of TNF-αand IL-1β. |
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