| ObjectiveBy observing the levels of autophagy and ROS of peripheral blood mononuclear cells in normal people,MDS patients with Right-Vaculty pattern and those with Stasis-Toxin pattern as well as the effect on the levels of autophagy and ROS in the MDS cell lines SKM-1 intervened in with Sanzihuangshi powder,a self-made preparation,to prove that:?the levels of autophagy and ROS are different among normal group,MDS patients with Right-Vaculty pattern and those with Stasis-Toxin pattern by extracting the PBMC for relevant detection.?the MDS cell line SKM-1 intervened in with Sanzihuangshi powder,a self-made preparation,can increase the autophagy level and reduce the ROS level.?the proposed hypothesis:autophagy is a form of Qihua,and MDS patients with Right-Vaculty are at the stage of Qihua mildly weakened,where Qihua can preliminary accumulate in the body metabolism stasis poison and steady condition.And MDS patients with Stasis-Toxin are at the stage of Qihua intensively weakened,where Qihua ability is not enough to build up in the body metabolism stasis poison and rapid deterioration.MethodsIn this study,20 cases of patients with MDS with Right-Vaculty pattern and those with Stasis-Toxin pattern and 20 cases of normal people who met the requirements were collected.Three groups of peripheral blood mononuclear cells were extracted.The number of autophagosomes in the three groups was observed by immunofluorescence microscopy.Flow cytometry was used to detect the fluorescence intensity of autophagy in the three groups.Western blot was used to detect the expression levels of autophagy related protein LC3and Beclinl and autophagy substrate protein p62.ROS fluorescence intensity of the three groups of cells was measured by a microplate assay.SPSS 21.0 statistical software was used for statistical analysis of the experimental results.At the same time,the MDS cell line SKM-1 was selected in this study and divided into the dosing group and the blank control group for simultaneous culture.The dosing group was intervened with self-made fuzheng detoxification compound Sanzihuangshi powder for 24 hours.The number of autophagosomes in the three groups was observed by immunofluorescence microscopy.Flow cytometry was used to detect the fluorescence intensity of autophagy in the three groups.Western blot was used to detect the expression levels of autophagy related protein LC3and Beclinl and autophagy substrate protein p62.ROS fluorescence intensity of the three groups of cells was measured by a microplate assay.SPSS 21.0 statistical software was used for statistical analysis of the experimental results.ResultsPart ?(1)Flow cytometry showed that the autophagy level of PBMC in the right-vaculty group was higher than that in the normal group(P<0.05);the autophagy level of PBMC in the stasis-toxin group was lower than that in the normal group(P<0.05);the autophagy level of PBMC in the stasis-toxin group was significantly lower than that in the right-vaculty group(P<0.001).(2)Western blotting showed that the p62 expression level of PBMC in the right-vaculty group was lower than that in the normal group(P<0.05);the p62 expression level of PBMC in the stasis-toxin group was higher than that in normal group(P<0.05);the p62 expression level of PBMC in the stasis-toxin group was significantly higher than that in the right-vaculty group(P<0.001);the beclin1 expression level of PBMC in the right-vaculty group was higher than that in the normal group(P<0.05);the beclin1 expression level of PBMC in the stasis-toxin group was lower than that in the normal group(P<0.05);the beclin1 expression level of PBMC in the stasis-toxin group was lower than that in the right-vaculty group(P<0.05);the LC3? expression level of PBMC in the right-vaculty group was higher than that in the normal group(P<0.05);the LC3?expression level of PBMC in the stasis-toxin group was lower than that in normal group(P<0.05);the LC3? expression level of PBMC in the stasis-toxin group was significantly lower than that in the right-vaculty group(P<0.001).(3)ROS test showed that the ROS level of PBMC in the right-vaculty group was significantly higher than that in the normal group(P<0.001);the ROS level of PBMC in the stasis-toxin group was significantly higher than that in the normal group(P<0.001);there is no statistical difference between the ROS level of PBMC in the stasis-toxin group and that in the right-vaculty group(P>0.05).Part ?(1)Flow cytometry showed that the autophagy level of SKM-1 in the dosing group was higher than that in the control group(P<0.05).(2)Western blotting showed that the p62 expression level of SKM-1 in the dosing group was lower than that in the control group(P<0.05);the beclin1 expression level was higher than that in the control group(P<0.05);the LC3II expression level was higher than that in the control group(P<0.05).(3)ROS test showed that the ROS level of SKM-1 in the dosing group was significantly lower than that in the control group(P<0.001).Conclusions(1)Different patterns in patients with MDS autophagy level and the level of ROS was toxic metabolites vary:in patients with MDS with Right-Vaculty,autophagy and ROS levels were higher than those in normal group.In patients with MDS with Stasis-Toxin,autophagy level is lower than that in normal group,and ROS level is higher than that in Right-Vaculty group and the normal group.(2)In PBMC of MDS patients with Right-Vaculty,The increase of autophagy is related to the preliminary accumulation of ROS.And PBMC of MDS patients with Right-Vaculty still has the ability to compete with gradually accumulated ROS by increasing autophagy.The decrease of PBMC autophagy in MDS patients with Stasis-Toxin is related to the excessive accumulation of ROS.And the autophagy ability of PBMC in MDS patients with Stasis Toxin is absolutely insufficient to compete with the excessive accumulation of ROS.(3)Combined with traditional Chinese medicine Qihua theory to explain the MDS syndrome and pathogenesis,MDS patients with Right-Vaculty are at the stage of Qihua mildly weakened,where Qihua can preliminary accumulate in the body metabolism stasis poison and steady condition.And MDS patients with Stasis-Toxin are at the stage of Qihua intensively weakened,where Qihua ability is not enough to build up in the body metabolism stasis poison and rapid deterioration.(4)Fuzheng detoxification compound Sanzihuangshi powder with the function of regulating Qihua can reduce ROS by increasing the autophagy amount of MDS cell line SKM-1,which verifies the hypothesis that autophagy is a form of Qihua,and fuzheng detoxification compound can regulate the level of autophagy by enhancing the Qihua function. |
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