| Objective:Through clinical observation and experimental research,we explored the clinical efficacy of Jianpi Xiaoke Recipe in patients with type2 diabetes mellitus with spleen deficiency and blood stasis and the regulation of the morphology and function of isletβcells through mTOR/SAD-A signaling pathway.Methods:Clinical study:A total of 64 patients with type 2 diabetes were enrolled in the study.They were randomly divided into the experimental group and the control group by 32 patients.The control group was treated with basal therapy+glimepiride（imoliol）.The experimental group was the control group of the control group+Jianpi Xiaoke Recipe.Both groups were treated for 8weeks.The changes of clinical symptoms,blood glucose,glycosylated hemoglobin,insulin levels,and isletβcell function index were observed before and after treatment.Experimental study:The islet cell strain was cultured in a high-sugar（25mmol/L）environment,and the The Jianpi Xiaoke Recipe extractant was diluted into different concentration gradients to intervene in high glucose cultured islet cells.CCK-8（Cell Counting Kit-8）method was used to detect the survival rate of islet cells,and the optimal concentration and optimal time of Jianpi Xiaoke Recipe were screened.Grouping:Low glucose control group（LG group）,high glucose model group（HG group）,Jianpi Xiaoke Recipe group（J group）,leucine group（LA group）,leucine+the best concentration group of Jianpi Xiaoke Recipe（LA+J group）,rapamycin group（LP group）,rapamycin+the best concentration group of Jianpi Xiaoke Recipe（LP+J group）,glucose-stimulated insulin secretion release test(glucose-Stimulated insulin secretion（GSIS）was used to detect the insulin concentration in the cell culture supernatants of each group.After routine,activation and blocking of mTOR,the m RNA activities of mTOR and SAD-A were measured,and the protein expression levels of mTOR and SAD-A were detected.Results:1.Clinical study results:After the study,2 patients in the control group and the test group were detached,and 30 patients in each group completed the test.Compared with the control group,the FPG,PBG and Hb A1c of the test group and the control group were compared.There were decreased（P<0.05）,FINS,2h INS,HOMA-βincreased（P<0.05）,and the total scores of TCM syndromes of spleen deficiency and blood stasis decreased（P<0.05）.After treatment,there was no significant difference in the decrease of FPG,PBG and FINS in the experimental group compared with the control group（P>0.05）.The decrease of Hb A1c,2h INS and HOMA-βin the experimental group was significantly different from that in the control group（P<0.05）.The TCM syndrome scores of the experimental group were significantly different from those of the control group（P<0.05）.The efficacy of the test group in improving the test indicators and TCM symptom scores was significantly better than that of the control group,suggesting that Jianpi Xiaoke Recipe has significant advantages in improving blood sugar,islet cell function and clinical symptoms.2.Experimental results:The cell viability was at the peak when the concentration of traditional Chinese medicine reached 10-3mg/m L,which was significantly higher than that of other traditional Chinese medicine concentration group and high glucose-free Chinese medicine group（P<0.05）,suggesting that the optimal concentration of traditional Chinese medicine is 10-3mg/m L.3.Compared with LG group,the cell viability of HG group was significantly decreased（P<0.05）.Compared with HG group,the activity of MIN6 cells in group J and LA group were significantly increased（P<0.05）;compared with LA group,the activity of MIN6 cells in LA+J group was better than that in LA group（P<0.05）.Compared with HG group,the cell viability of LP group was significantly lower（P<0.05）.Compared with LP group,LP+J group was better than LP group（P<0.05）,suggesting that high glucose can lead to decreased cell viability,while Jianpi Xiaoke Recipe and leucine can alleviate the decrease of cell viability caused by high glucose environment,and the two can synergistically enhance the action.Rapamycin inhibits cell viability,and Jianpi Xiaoke Recipe is consistent with leucine,which activates mTOR and enhances cell viability.4.Compared with the LG group,the insulin level in the HG group was significantly decreased（P<0.05）.Compared with the HG group,the insulin levels in the J group and the LA group were significantly higher than those in the HG group（P<0.05）,compared with the LA group,the insulin secretion level of LA+J group was higher than that of LA group（P<0.05）.Compared with HG group,the insulin level of LP group was significantly decreased（P<0.05）.Compared with LP group,the insulin level of LP+J group was higher than that of LP group（P<0.05）.Indicating that high glucose environment will cause the GSIS level to be significantly reduced,while Jianpi Xiaoke Recipe and leucine can improve the GSIS inhibition caused by high glucose,synergistically enhance the effect,rapamycin can inhibit the insulin secretion of cells,and Jianpi Xiaoke Recipe has the same mechanism of action as leucine,which can activate mTOR and improve GSIS.5.In terms of the expression of mTOR protein,compared with LG group,the relative expression level of mTOR protein in HG group was decreased（P<0.05）;compared with HG group,the expression levels of mTOR protein in group J and LA group were significantly increased（P<0.05）;compared with LA group,the relative expression level of mTOR protein in LA+J group was increased（P<0.05）;compared with HG group,the relative expression level of mTOR protein in LP group was significantly lower（P<0.05）;compared with LP group,the relative expression level of mTOR protein in LP+J group was increased（P<0.05）.In terms of SAD-A protein expression,compared with LG group,the relative expression level of SAD-A protein in HG group was decreased（P<0.05）;compared with HG group,the relative expression levels of SAD-A protein in LA group and J group were also higher than those in HG group（P<0.05）;compared with LA group,the relative expression level of SAD-A protein in LA+J group was higher than that in LA group（P<0.05）;compared with HG group,the relative expression level of SAD-A protein in LP group was significantly lower than that in HG group（P<0.05）;compared with the LP group,the relative expression level of SAD-A protein in LP+J group was higher than that in LP group（P<0.05）.It is suggested that high glucose environment can inhibit the expression of mTOR and SAD-A protein,and activate the expression of mTOR protein,which can also increase the expression of SAD-A protein,both Jianpi Xiaoke Recipe and leucine can increase the expression of mTOR and SAD-A protein,and they can synergistically enhance protein expression,rapamycin inhibits the expression of SAD-A protein while inhibiting mTOR,suggesting that Jianpi Xiaoke Recipe can alleviate the inhibitory effect of rapamycin on the expression of mTOR and SAD-A protein.6.Compared with LG group,the relative expression levels of mTOR and SAD-A m RNA in HG group were lower（P<0.05）;compared with HG group,the relative expression levels of mTOR and SAD-A m RNA in group J and LA group were significantly increased（P<0.05）;compared with LA group,the relative expression levels of mTOR and SAD-A m RNA in LA+J group were higher than those in LA group（P<0.05）;compared with the HG group,the relative expression levels of mTOR and SAD-A m RNA in the LP group were significantly lower（P<0.05）;compared with the LP group,the relative expression levels of mTOR and SAD-A m RNA in the LP+J group were increased（P<0.05）.It is suggested that high glucose environment can inhibit the expression of mTOR and SAD-A m RNA,and activate the expression of mTOR m RNA,which can also increase the expression of SAD-A m RNA,both Jianpi Xiaoke Recipe and leucine can increase the expression of mTOR and SAD-A m RNA,and synergistically enhance expression,rapamycin inhibits the expression of SAD-A m RNA while inhibiting the expression of mTOR m RNA,while Jianpi Xiaoke Recipe can alleviate the inhibitory effect of rapamycin on mTOR and SAD-A m RNA.Conclusion:Jianpi Xiaoke Recipe can improve the TCM symptoms of spleen deficiency and blood stasis type 2 diabetes patients and increase the isletβcell function index;its mechanism may be related to its ability to improve isletβcell function by activating mTOR/SAD-A signaling pathway. |
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