| Purpose To explore the protective effect of Yiqi Jiedu Active Herbal Medicine on the damage of human glomerular mesangial cells,and to elucidate the therapeutic mechanism of Yiqi Jiedu Active Herbal Medicine in the prevention and treatment of diabetic nephropathy,and to provide new ideas for the clinical prevention and treatment of diabetic nephropathy.The future development of new drugs to provide theoretical basis.Methods1.Cell source Human glomerular mesangial cell line(HBZY-1)gift from Liaoning University of Traditional Chinese Medicine Laboratory of Basic Medicine2.Experimental drug Astragaloside : product batch number 201314,Liaoning North Pharmaceutical Technology Development Co.,Ltd.Berberine Hydrochloride: Product Lot 15010411,Beijing Yingze Na New Chemical Technology Research Institute Hirudin: product batch number V0145,gracia(Chengdu)Chemical Technology Co.,Ltd.Lilacoside: product batch number 15012204,purchased from Beijing Ying Ze Na new chemical technology research institute3.Cell culture Human mesenchymal stem cells(GMC)were treated with a pipette.The cells were resuspended in a 100 ml culture flask.The complete culture medium(DMEM +10m L / d L fetal bovine serum containing 100 U / m L blue,streptomycin)The(80%),with the pipette aspiration of the culture medium,adding 0.25% trypsin digestion for3 minutes,inverted microscope to observe the cell gradually fall off,add the cell to the cell growth state,DMEM(low sugar complete medium)solution to terminate the digestion,with a pipette gently blow to the suspension state,the cell suspension into the 15 m L centrifuge tube,1200 rpm centrifugal 10 minutes,to the supernatant,add DMEM(low sugar completely Medium)to adjust the cell concentration to 1 × 105 /m L,1: 3 ratio inoculated in 25 m L cell culture flask The flask into 5% CO2,37 ℃incubator,every 2d pass 1 times,about passaged 4 times after the start of the test.4.Experimental grouping1.In the experimental one,two,three of the human glomerular mesangial cells by 4factors 3 level orthogonal design program is divided into the following 11 groups:Normal group(5.6 m M glucose)High glucose and high fat model group(30m M glucose + 20 mg / L LPC)Group 1: Berberine Hydrochloride(Low)Astragaloside(Low)Hirudin(Low)Luteolin(Low)Group 2: Berberine Hydrochloride(Low)Astragaloside(Medium)Hirudin(Medium)Luteolin(Medium)Group 3: Berberine Hydrochloride(Low)Astragaloside(H)Hirudin(High)Luteolin(H)Group 4: Berberine Hydrochloride(Medium)Astragaloside(Low)Hirudin(Medium)Luteolin(H)Group 5: Berberine Hydrochloride(Medium)Astragaloside(Medium)Hirudin(High)Luteolin(Low)Group 6: Berberine Hydrochloride(Medium)Astragaloside(H)Hirudin(Low)Luteolin(Medium)Group 7: Berberine Hydrochloride(High)Astragaloside(Low)Hirudin(High)Luteolin(Medium)Group 8: Berberine Hydrochloride(H)Astragaloside(Medium)Hirudin(Low)Luteolin(H)Group 9: Berberine Hydrochloride(H)Astragaloside(Low)Hirudin(Medium)Luteolin(H) Berberine Hydrochloride is low,medium and high doses were 20μM,180μM;Astragaloside low,medium and high doses were 20μM,60μM,180μM;Hirudin low,medium and high doses were 0.5μM,1.5μM,4.5μM;Luteolin low,medium and high doses were 60μM,180μM,540μM.2.According to the experimental one,two,three analysis results,select the best combination of Qi and detoxification active Chinese medicine program for experimental four,are grouped as follows:Normal group(5.6 m M glucose)High glucose and high fat model group(30m M glucose + 20 mg / L LPC)Group 1: Berberine Hydrochloride(Low)Astragaloside(Low)Hirudin(Low)Luteolin(H)Group II: Berberine Hydrochloride(Low)Astragaloside(Low)Hirudin(Low)Luteolin(Low)Group 3: Berberine Hydrochloride(Low)Astragaloside(Medium)Hirudin(Medium)Luteolin(Medium)Berberine hydrochloride low,medium and high doses were 20μM,60μM,180μM;Astragaloside low,medium and high dose were 20μM,60μM,180μM;Hirudin low,medium and high dose were 0.5μM,1.5μM,4.5μM;Luteolin low,medium and high doses were 60μM,180μM,540μM.5.Index detection Experiment 1:MTT assay was used to detect the proliferation of GMC at 24 h,48h and 72 h.Experiment 2:The m RNA expression of FN and COLIV 24 and 48 h were detected by q RT-PCR The expression of FN,COLIV and AGEs was detected by ELISA Experiment 3:The m RNA expression of TGF-β1 and MCP-1 24 and 48 h were detected by q RT-PCR The expression of TGF-β1 and MCP-1 48 h was detected by ELISA Experiment 4:The m RNA expression of PPAR-γ 24 and 48 h was detected by q RT-PCR The expression of PPAR-γ 24 and 48 h protein was detected by Western Blot Western blot was used to detect the expression of NF-κB protein in high glucose and high fat model group at 0,15,30,60,90,120 min,and the highest time point of NF-κB expression was selected.The expression of NF-κB protein was detected by Western blotting Nuclear transfection was used to detect NF-κB nuclear transfection in each group.6.Data processing SPSS17.0 statistical software was used to process the experimental data.All the data were expressed as ± s.The data of each group were analyzed by orthogonal design.The mean number of each group was compared with LSD method,P <0.05 was statistically significant.Result1.Effects of compatibility of traditional Chinese medicine on the proliferation of GMC cells induced by high glucose and high fat MTT method was used to observe the changes of GMC proliferation at each time point:Compared with the model group,the proliferation of GMC cells was significantly decreased at 24,48 and 72 hours(P <0.01).Compared with the normal control group,the proliferation of GMC cells in the model group was significantly different(P <0.01).There was no significant difference between the two groups(P> 0.05).There was significant difference between the other groups(P <0.01).There was no significant difference between the two groups(P> 0.05).There was significant difference between the other groups(P <0.01).There was no significant difference between the two groups at the time of 72h(P> 0.05).Comparison of traditional Chinese medicine group found: The proliferation of GMC cells was significantly decreased at 7,9 group(P <0.01).At 48 h,the proliferation of GMC cells decreased significantly in6、7、8 group(P <0.01).There was significant difference between 5and 7 group(P <0.05).There was no significant difference between the other groups(P<0.01).24 h,48h,72 h three time points compared to normal group 48 h cell proliferation higher than 24 h and 72h(P <0.01),the proliferation of the model group 72 h was higher than that of the 24 h and 48h(P <0.01),the proliferation of 48 h cells was higher than that of 24 h and 72 h(P <0.01).2.Effect of Yiqi Jiedu Active Herbal Medicine on Expression of FN m RNA in GMC Cells Induced by High Glucose and High Fat24h: Compared with the normal group,except for one group,the model group and the compatibility of traditional Chinese medicine group FN m RNA changes were significantly different(P <0.01);Compared with the model control group,the relative expression of FN m RNA in the traditional Chinese medicine group was significantly decreased(P <0.01);The expression of FN m RNA was significantly improved in 1 and 2 groups(P <0.01).48h: Compared with the normal group,there were significant differences in FN m RNA between the model group and the traditional Chinese medicine group(P <0.01).Compared with the model control group,the relative expression of FN m RNA in Chinese herbal medicine group was significantly decreased(P <0.01).The expression of FN m RNA was significantly improved in 1 and 2 groups(P <0.01).There was no significant difference in FN m RNA expression between the normal group and the model group at 24 and 48 hours(P> 0.05).The expression of FN m RNA in group 1and group 2 was lower than that in 24 h group(P <0.01).3.Effect of Fuqi Jiedu Active Herbal Medicine on Expression of FN Protein in GMC48 h Induced by High Glucose and High Fat Compared with the normal control group,the model control group and the compatibility of traditional Chinese medicine group were significantly increased(P <0.01);Compared with the model group,the expression of FN protein in the traditional Chinese medicine group was significantly lower than that in the control group;Compared with the orthogonal group,the expression of FN protein in the first group was the highest(P <0.01).4.Effect of Yiqi Jiedu Active Herbal Medicine on COLIV m RNA Expression in GMC Cells Induced by High Glucose and High Fat24h: Compared with the normal group,the changes of COLIV m RNA in the model group and the Chinese herbal medicine group were significantly different(P <0.01),Compared with the model group,the relative expression of COLIV m RNA was significantly decreased(P <0.01),There was no significant difference between the 2 group and the 3 group and the 6 group and the 8 group(P> 0.05),Among them,COLIV m RNA was the most significant(P <0.01).48h: Compared with the normal group,the changes of COLIV m RNA in model group and Chinese herbal medicine group were significantly different(P <0.01,group 1,P <0.05),Compared with the model group,the relative expression of COLIV m RNA in Chinese herbal medicine group was significantly decreased(P <0.01),There was no significant difference between the 2 and 3 groups(P> 0.05),And COLIV m RNA was the most significant(P<0.01).There was no significant difference in Col IV m RNA expression between normal group,model group and traditional Chinese medicine group at 24 and 48 hours(P> 0.05).The expression of FN m RNA in the 2 and 3 groups was lower than that in the control group(P<0.01).5.Effects of Combination of Chinese Herbal Medicine on the Expression of COLIV Protein in GMC Cells Induced by High-glucose and High-fat Compared with the normal group,there was no significant difference(P> 0.05),except the model control group and the traditional Chinese medicine group(P <0.01).Compared with the model group,the expression of COl IV protein was significantly down-regulated compared with the traditional Chinese medicine group.Compared with the traditional Chinese medicine group,the expression of COl IV protein was down-regulated in one group(P <0.01). 6.Effect of Yiqi Jiedu Active Herbal Medicine on the Expression of AGEs Protein in GMC Cells Induced by High Glucose and High Fat Compared with the normal group,the expression of AGEs protein in the model group and the traditional Chinese medicine group was significantly higher than that in the control group(P <0.01).Compared with the model group,the expression of AGEs protein was significantly decreased(P <0.01).Compared with the other groups,the activity of AGEs protein in group 1 was better than that in other groups(P <0.01).7.Effect of Yiqi Jiedu Active Herbal Medicine on Expression of TGF-β1 m RNA in GMC Cells Induced by High Glucose and High Fat Compared with the normal control group,the changes of TGF-β1 m RNA were significantly different between the model group and the Chinese herbal medicine group at 24 and 48 hours(P <0.01).Compared with the model control group,the relative expression of TGF-β1 m RNA in the Chinese herbal medicine group was significantly decreased at 24 and48 hours(P <0.01).Compared with the two groups,the expression of TGF-β1 m RNA was significantly improved in 24 and 48 hours(P <0.01).There was no significant difference in TGF-β1 m RNA expression between the normal group and the model group at 24 and 48 hours(P> 0.05).The expression of TGF-β1 m RNA in the two groups was significantly lower than that in the control group(P <0.01).8.Effect of Yiqi Jiedu Active Herbal Medicine on Expression of TGF-β1 Protein in GMC Cells Induced by High Glucose and High Fat Compared with the normal group,the expression of TGF-β1 protein in the model group and the traditional Chinese medicine group increased significantly(P <0.01).Compared with the model group,the expression of TGF-β1 protein in the Chinese herbal medicine group was significantly down-regulated(P <0.01).Compared with the traditional Chinese medicine group,the expression of TGF-β1 protein was the best in the 2 group(P <0.01).9.Effect of Yiqi Jiedu Active Herbal Medicine on MCP-1 m RNA Expression in GMC Cells Induced by High Glucose and High Fat24h: compared with the normal group,except one group(P> 0.05),There was significant difference between the model group and the other TCM groups(P <0.01).Compared with the model group,the relative expression of MCP-1 m RNA in the compatibility group was significantly decreased(P <0.01).Compared with the traditional Chinese medicine group,the expression of MCP-1 m RNA was the most significant(P <0.01).48h: Compared with the normal group,there was significant difference between the model group and the traditional Chinese medicine group(P <0.01),Compared with the model group,the relative expression of MCP-1 m RNA was significantly decreased(P <0.01),Compared with the traditional Chinese medicine group,the decrease of MCP-1 m RNA was the most significant(P <0.01).There was no significant difference in MCP-1 m RNA expression between the normal group and the model group at 24 and 48 hours(P> 0.05).The expression of MCP-1 m RNA was significantly lower than that of 24h(P <0.05).10.Effects of high glucose and high fat environment on the expression of NF-κB protein in GMC cells The expression of NF-κB protein in the high glucose and high fat model group was detected by Western blot,and the expression of NF-κB was the most significant in the 90 th day at the time of 90 min,90,120 and 120 min.11.Effects of Yiqi Jiedu Active Herbal Medicine on NF-κB Protein Expression in GMC Cells under High Glucose and Lipid Environment Compared with the normal group,the expression level of NF-κB protein was significantly higher in the model group at 60 min and 90 min than that in the control group at60 min,and the expression of NF-κB protein was down-regulated in the first and third groups at 90 min The most significant level.12.Effects of Compatibility of Chinese Herbal Medicine on the Changes of NF-κB Nuclear Transfection in High-glucose and High-fat Environment Compared with the normal group,the NF-κB subunit p65 of the model group was mainly distributed in the nucleus at 60 min and 90 min,and the p65 of the normal group and the traditional Chinese medicine group were mostly distributed outside the nucleus.Conclusion1.(FN),type 4 collagen(COLIV),and the proliferation of human glomerular mesangial cells(GMC)induced by high glucose and high fat in vitro,and to inhibit the proliferation of human glomerular mesangial cells(GMC)MRNA and protein levels,and down-regulate the expression of advanced glycation end products(AGEs).It is suggested that the combination of active and detoxifying traditional Chinese medicine can inhibit the glomerular arteriosclerosis by inhibiting GMC extracellular matrix and AGES synthesis.2.The expression of TGF-β1,MCP-1 and NF-κB in GMC cells can inhibit the expression of PPAR-γ in GMC cells.It is suggested that the compatibility of traditional Chinese medicine components can play a role in preventing and treating DN by regulating the expression of related cytokines and signaling pathways.3.The possible combination of the active ingredients of active and active Chinese medicine may protect the damaged GMC by inhibiting the expression of MCP-1,FN,COLIV and AGEs up-regulated the expression of PPAR-γ by inhibiting the TGF-β1 / NF-κB signaling pathway To slow the progress of DN disease,to protect the role of damaged kidneys.4.The best compatibility formula for the compatibility of traditional Chinese medicine is Berberine Hydrochloride(Low Dose 20μM)Astragaloside(Low Dose 20μM)Hirudin(Low Dose 0.5μM)Luteolin(high dose of 540μM). |
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