| Objective To observe the protective effect of phlegm and blood stasis treatment on high glucose-induced injury of cardiac microvascular endothelial cells(CMECs),to explore the mechanism of intervening diabetic myocardial microangiopathy by regulating AGEs-RAGE signal pathway,and to reveal the treatment of phlegm and blood stasis to prevent diabetes Molecular Mechanisms of Myocardial Microangiopathy.Methods 1.Isolation,culture and identification of rat CMECs One SD rat was selected.Under sterile conditions,the free wall of the left ventricle of the rat was isolated by digestion and cultured in the incubator.Rat CMECs were passaged for identification after identification according to the characteristics of the cells.2.Preparation of rat drug-containing serum and empty pack control serum Sixty male SD rats were selected for adaptive feeding for 3 days.Forty of them,10 rats in each group were dosed with 4.05 g / kg,4.05 g / kg,8.10 g / kg,and 0.3 g / kg of intragastric appetizing soup,small trapped chest soup,resisting trapped chest soup and ALT-711 solution was prepared into drug-containing serum(dose was administered at 10 times the daily routine daily dose in vitro);another 20 rats were perfused with an equal volume of distilled water to prepare a blank control serum.Intragastric administration was performed twice a day for 7 days.Rats were anesthetized 1 h after the last gastric lavage,and blood was taken from the abdominal aorta.The rats were left at room temperature for 2 h,centrifuged at 3000 r / min for 15 min to obtain the supernatant,and the complement was inactivated at 56 ℃ for 30 min.The microporous filter was used for sterilization,and the serum was divided into 5m L EP tubes.The refrigerator was stored at-80℃ for later use.3.Experimental grouping and intervention CMECs in logarithmic growth phase were taken,digested,centrifuged,and the concentration was adjusted to 1×105/ml,and cells were seeded on 6-well plates.Set up 6 groups:blank control(NC)group(blank control serum),model(MC)group(glucose+blank control serum),Huayu(DBS)group(glucose+Daotang decoction-containing serum),and phlegm(RP)group(glucose+Xiaoxang chest soup with medicated serum),phlegm and blood stasis treatment(RPDBS)group(glucose+Dadang depression chest with medicated serum)and western medicine(ALT-711)group(glucose+ALT-711 decoction with medicine serum).The cells were collected in a 37℃,5%CO2 incubator,and the detection indicators were as follows:(1)Simultaneous treatment of phlegm and stasis protects CMECs with high glucose damage by regulating the AGEs-RAGE signal axis(1)MTT method to select the best glucose concentration;(2)MTT method to screen the best percentage of medicated serum to protect model cells;(3)ELISA method was used to detect the content of AGEs in each group of cells;(4)Immunofluorescence,Western blot and real-time quantitative PCR were used to detect the expression of RAGE protein and gene level in cells;(2)AGEs-RAGE signal axis activates oxidative stress to interfere with detection of CMECs lesions(1)Cytochrome c reduction method to detect NADPH oxidase activity;(2)Ethyl dihydrogen(DHE)fluorescent probe method was used to detect ROS level.Results(1)After identification,rat CMECs were successfully isolated and cultured in vitro.(2)The phlegm and blood stasis treatment method protects CMECs with high glucose damage by inhibiting AGEs-RAGE signal axis over-activation(1)Screening of the optimal glucose modelling concentration by MTT method:After glucose concentration is 33 mmol/L,after 48 hours of intervention,the viability of modelling cells drops to about 50%.Considering a comprehensive selection of 33 mmol/L glucose,48 hours of intervention Optimal conditions for high glucose-induced myocardial microvascular endothelial cell modeling.(2)Screening of the optimal percentage of model-containing cells protected by drug-containing serum by MTT method: The viability was best when the percentage was 10%,and the drug-containing serum of Dangzhan Decoction interfered with model cells for 48 h.(3)AGEs were detected by ELISA method: MC group had significantly higher intracellular AGEs content than NC group(P <0.01);AGEs content of each medication group was significantly lower than MC group(P <0.01),among which RPDBS group was more than RP group and DBS.Group,the AGEs content decreased significantly(P <0.01),the RBDBS group and the ALT-711 group had a similar effect in reducing the AGEs content(P> 0.05).(4)Immunofluorescence,Western blot and real-time fluorescence quantitative detection of RAGE protein level and gene level expression in endothelial cells: Compared with NC group,the positive protein expression of endothelial cells in MC group increased(P <0.01),and gene expression level increased.(P <0.01);Compared with the MC group,the protein and gene expression of each drug group was significantly reduced(P <0.01);among them,the RAGE protein level and gene level were significantly reduced in the RPDBS group and the RP group(P <0.01,P <0.05).(3)AGEs-RAGE signal axis inhibits oxidative stress and prevents CMECs lesions(1)Detection of NADPH oxidase activity by cytochrome c reduction method: Compared with the NC group,the NADPH oxidase activity level of the endothelial cells increased in the MC group(P <0.01);compared with the MC group,the NADPH oxidase activity level of each drug group was significantly reduced.(P <0.01).The reduction of NADPH oxidase activity in the RPDBS group and the ALT-711 group was similar(P> 0.05).Compared with the RPDBS group,the reduction of NADPH oxidase activity was statistically significant(P <0.01).(2)Detection of ROS levels in endothelial cells by fluorescent probe method: Compared with the NC group,the ROS level in the MC group was significantly increased(P<0.01);compared with the MC group,the ROS level in each medication group was significantly reduced(P<0.01),of which RPDBS The effect of reducing ROS levels was similar between the ALT-711 group and the ALT-711 group(P>0.05).Compared with the RPDBS group and the RP group,the reduction of ROS level was statistically significant(P<0.01).Compared with the RPDBS group and the DBS group,the ROS reduction level was not statistically significant.Significance(P>0.05).Conclusion The phlegm and blood stasis treatment method may interfere with diabetic myocardial microangiopathy by inhibiting the excessive activation of AGEs-RAGE signal axis and inhibiting oxidative stress response to protect CMECs with high glucose damage. |
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