| 1.ObjectiveTo investigate the mechanism of systemic lupus erythematosus(SLE)mesangial cell proliferation mediated by lncRNA GAS5/ miR-21-5p/ Sprouty1 from the perspective of ce RNA using in vitro cell and animal model experiments.To reveal the efficacy of Qihuang Jianpi Zishen Granules(QJZ)in renal damage of MRL/lpr mice and the regulation of lncRNA GAS5/ miR-21-5p/ Sprouty1.To elucidate the molecular mechanism of QJZ to improve kidney damage in SLE.2.Methods2.1 Research 1: Study on the mechanism of the combination of lncRNA GAS5/miR-21-5p/Sprouty1 regulating ERK/CREB pathway mediating SLE mesangial cell proliferationIn this study,Human mesangial cells(HMCs)were used as the model to stimulate HMC in vitro with TGF-β1.Based on gene transfection techniques,the functional experiments of lncRNA GAS5/miR-21-5p/Sprouty1 combination have been constructed with gene overexpression plasmid and small interfering RNA(si RNA).Dual luciferase reporting assay verified the targeting binding relationship between lncRNA GAS5,miR-21-5p and Sprouty1.A series of reverse experiments were conducted to verify the regulatory relationship between lncRNA GAS5,miR-21-5p and Sprouty1 and their effects on HMC cell proliferation.CCK-8 was used to detect HMC cell viability.The cell cycle of HMC was detected by FCM.The contents of IL-1,IL-6 and TNF-α in the supernatant were determined by ELISA.lncRNA GAS5/ miR-21-5p/ Sprouty1 combination and the expression of ERK/CREB signaling pathway were detected by RT-q PCR,WB and IF.2.2 Research 2: The efficacy of Qihuang Jianpi Zishen Granules on kidney damage in MRL/lpr mice and its influence on lncRNA GAS5/ miR-21-5p/ Sprouty1combinationIn this study,MRL/lpr mice were used as the animal model of SLE.Forty 8-week-old female MRL/lpr mice were randomly divided into model group and Prednisone group(Pred),QJZ group,Mycophenolate mofetil(MMF)group,there were 10 mice in each group.Ten 8-week-old female C57BL/6 mice were used as control group(Conrtol).Pred group mice were treated with Pred;Mice in QJZ group were treated with QJZ based on Pred;Mice in MMF group were treated with MMF based on Pred;After 8weeks of continuous medication,samples were collected for detection.Serum Scr,BUN,cytokine IL-1,IL-6,TNF-α,anti-DSDNA antibody,C3,C4 were detected by ELISA.BCA determination of urinary protein;Renal pathology was observed by HE and Masson staining,and activity index(AI)and chronic index(CI)scores were performed.The expressions of lncRNA GAS5,miR-21-5p,Sprouty1,ERK1/2 and CREB in kidney tissues were detected by RT-q PCR.The expressions of Sprouty1,ERK1/2 and CREB proteins were detected by Western blotting.2.3 Research 3: Study on inhibition of SLE mesangial cells proliferation by regulating ERK/CREB pathway with the serum of Qihuang Jianpi Zishen Granules through lncRNA GAS5/miR-21-5p/Sprouty1In this study,HMC was used as the model.QJZ-containing serum was prepared and the effects of QJZ-containing serum on HMC cell proliferation,cytokines IL-1,IL-6 and TNF-α,lncRNA GAS5/miR-21-5p/Sprouty1 combination and ERK/CREB pathway were observed.On the basis of interfering the expression of lncRNA GAS5,to observe whether QJZ-containing serum can save the influence of si-GAS5 on HMC cell proliferation,cytokines IL-1,IL-6 and TNF-α,lncRNA GAS5/miR-21-5p/Sprouty1 combination and ERK/CREB pathway.It was verified that QJZ-containing serum inhibited HMC cell proliferation by regulating lncRNA GAS5/miR-21-5p/Sprouty1 combination.3.Results3.1 Research 1: Study on the mechanism of the combination of lncRNA GAS5/miR-21-5p/Sprouty1 regulating ERK/CREB pathway mediating SLE mesangial cell proliferation(1)Screening of the optimal concentration and time of TGF-β1 stimulation of HMC:According to the results of CCK-8 detection,we selected the concentration of 10 ng/ml TGF-β1 for 24 h as the optimal concentration and time.(2)Expression of lncRNA GAS5,miR-21-5p and Sprouty1 in HMC: The results of RT-q PCR and IF detection showed that the expression of lncRNA GAS5 and Sprouty1 in HMC was decreased(P < 0.01),while the expression of miR-21-5p was increased(P <0.01).(3)Transfection efficiency of lncRNA GAS5,miR-21-5p and Sprouty1 in HMC: The results of RT-q PCR showed that the expression of lncRNA GAS5 and Sprouty1 decreased after transfection of si-GAS5 and si-Sprouty1 into HMC(P < 0.01).The expressions of lncRNA GAS5 and Sprouty1 were increased after transfection into HMC with 3.1-GAS5 and 3.1-Sprouty1 respectively(P < 0.01).The expression of miR-21-5p was increased after transfection of miR-21-5p mimic into HMC(P < 0.01).After transfection with miR-21-5p inhibitor into HMC,the expression of miR-21-5p decreased(P < 0.01).No effect was found in all NC groups(P > 0.05).(4)Functional experiments of lncRNA GAS5,miR-21-5p and Sprouty1: After lncRNA GAS5 overexpression,HMC cell proliferation was inhibited(P < 0.01)and ERK/CREB pathway activation was inhibited(P < 0.01);The expression of miR-21-5p was decreased(P < 0.01),while the expression of Sprouty1 was increased(P < 0.01).After lncRNA GAS5 interfered with the expression,the result was reversed.Mi R-21-5p mimic promoted HMC cell proliferation(P < 0.01),activated ERK/CREB pathway(P <0.01),and down-regulated Sprouty1 expression(P < 0.01),miR-21-5p inhibitor results were reverse.After Sprouty1 overexpression,HMC cell proliferation was inhibited(P <0.01)and ERK/CREB pathway activation was inhibited(P < 0.01).When Sprouty1 interferes with expression,the result is reversed.(5)Verification of the targeted regulatory relationship between lncRNA GAS5,miR-21-5p and Sprouty1: Dual luciferase reporting experiment results showed that there were binding sites between lncRNA GAS5 and miR-21-5p,and between miR-21-5p and Sprouty1.Compared with NC mimic group,the luciferase activity of lncRNA GAS5-wt was significantly decreased in miR-21-5p group(P < 0.01),while the luciferase activity of lncRNA GAS5-mut was not significantly changed(P > 0.05).Compared with NC mimic,the Sprouty1-wt luciferase activity of miR-21-5p group was significantly decreased(P < 0.01),while the luciferase activity of Sprouty1-mut group had no significant changes(P > 0.05).Therefore,it indicates that lncRNA GAS5,miR-21-5p and Sprouty1 can be targeted and regulated,and a ce RNA regulatory relationship can be formed.(6)Recovery Experiment 1: On the basis of lncRNA GAS5 overexpression,si-Sprouty1 was transfected.The results showed that the proliferation of HMC was increased(P <0.01),the contents of cytokines IL-1,IL-6 and TNF-α were increased(P < 0.01),and the ERK/CREB pathway was activated(P < 0.01).si-Sprouty1 can reverse the influence of lncRNA GAS5 overexpression.(7)Recovery Experiment 2: After overexpression of lncRNA GAS5,the HMC were transfected with miR-21-5p mimic.The results showed that the proliferation of HMC was increased(P < 0.01),and the contents of cytokines IL-1,IL-6 and TNF-α were increased(P < 0.01),and the ERK/CREB pathway was activated(P < 0.01),miR-21-5p mimic could reverse the effect of lncRNA GAS5 overexpression.3.2 Research 2: The efficacy of Qihuang Jianpi Zishen Granules on kidney damage in MRL/lpr mice and its influence on lncRNA GAS5/ miR-21-5p/ Sprouty1combination(1)The effect of QJZ on renal pathological damage in MRL/lpr mice: Compared with model group,AI and CI scores in Pred,QJZ and MMF groups were decreased(P <0.01);Compared with Pred group,AI and CI scores in QJZ group were decreased(P <0.05);There was no significant difference in AI and CI scores between QJZ and MMF group(P > 0.05).(2)The effect of QJZ on renal function of MRL/lpr mice: Compared with model group,24 h PRO,ACR,Scr and BUN levels in Pred,QJZ and MMF groups were decreased(P< 0.05);Compared with Pred group,the 24 h PRO,ACR and Scr levels in QJZ group were decreased more significantly(P < 0.01),but BUN decreased no significant change(P > 0.05).Compared with MMF group,QJZ improved ACR and Scr as well as MMF group(P > 0.05),while MMF significantly reduced 24 h PRO and BUN(P < 0.05).(3)The effects of QJZ on cytokines IL-1,IL-6 and TNF-α in MRL/lpr mice: Compared with model group,the levels of IL-1,IL-6 and TNF-α in Pred,QJZ and MMF groups were decreased(P < 0.01);Compared with Pred group,the levels of IL-1,IL-6 and TNF-α in QJZ group were decreased(P < 0.01);There were no significant differences in the levels of IL-1,IL-6 and TNF-α between QJZ group and MMF group(P > 0.05).(4)The effect of QJZ on immunological indexes of MRL/lpr mice: Compared with model group,the levels of anti-ds DNA antibody in Pred,QJZ and MMF groups were decreased(P < 0.01),while the levels of C3 and C4 were increased(P < 0.01);Compared with Pred group,the level of anti-ds DNA antibody in QJZ group was decreased(P < 0.01),and the levels of C3 and C4 were increased(P < 0.01).There were no significant differences in the levels of anti-ds DNA antibody,C3 and C4 in QJZ group compared with MMF group(P > 0.05).(5)The effects of QJZ on the combination of GAS5/ miR-21-5p/ Sprouty1 in kidney of MRL/lpr mice: compared with model group,the expressions of GAS5 and Sprouty1 in Pred,QJZ and MMF groups were increased(P < 0.01),while the expressions of miR-21-5p were decreased(P < 0.01);Compared with Pred group,GAS5 and Sprouty1 expressions were increased and miR-21-5p expression was decreased in QJZ group(P <0.01).(6)The effect of QJZ on renal ERK/CREB pathway in MRL/lpr mice: Compared with model group,the phosphorylation levels of ERK1/2 and CREB in Pred,QJZ and MMF groups were decreased(P < 0.01);Compared with Pred group,the phosphorylation level of ERK/CREB pathway was decreased in QJZ group(P < 0.05).3.3 Research 3: Study on inhibition of SLE mesangial cells proliferation by regulating ERK/CREB pathway with the serum of Qihuang Jianpi Zishen Granules through lncRNA GAS5/miR-21-5p/Sprouty1(1)Screening of the optimal concentration and time of the action of QJZ-containing serum on HMC: CCK-8 test results showed that the concentration of QJZ-containing serum was 10%,and the effect of 24 h on the activity of HMC cells was better.(2)Effects of QJZ-containing serum on HMC proliferation,cell cycle,cytokines IL-1,IL-6 and TNF-α,ERK/CREB pathway,lncRNA GAS5/miR-21-5p/Sprouty1combination: The results showed that QJZ-containing serum could inhibit the proliferation of HMC,blocking the proliferation of HMC in G1 phase of the cell cycle.Decreased the levels of cytokines IL-1,IL-6 and TNF-α(P < 0.01);It can up-regulate the expression of lncRNA GAS5 and Sprouty1(P < 0.01),down-regulate the expression of miR-21-5p(P < 0.01),and inhibit the activation of ERK/CREB pathway(P < 0.01).(3)QJZ-containing serum rescues the effects of si-GAS5 on HMC cell proliferation,cytokines IL-1,IL-6 and TNF-α,ERK/CREB pathway,lncRNA GAS5/miR-21-5p/Sprouty1 combination: The transfection of si-GAS5 was followed by the intervention of QJZ-containing serum.The results showed that the proliferation of HMC was inhibited,the contents of cytokines IL-1,IL-6 and TNF-α were decreased(P< 0.01),and the activation of ERK/CREB pathway was inhibited(P < 0.01).The expressions of lncRNA GAS5 and Sprouty1 were increased(P < 0.01),while the expression of miR-21-5p was decreased(P < 0.01).QJZ-containing serum could save the influence of lncRNA GAS5 interference expression.4.Conclusions(1)lncRNA GAS5/miR-21-5p/Sprouty1 can form ce RNA,and lncRNA GAS5 competitively binds to miR-21-5p,downregulates the expression of Sprouty1,regulates the ERK/CREB pathway,and mediates the proliferation of SLE mesangial cells.(2)Qihuang Jianpi Zishen granules could improve the pathological injury of kidney,the levels of urinary protein,renal function indexes,the contents of serum cytokines IL-1,IL-6 and TNF-α,and immunological indexes of MRL/lpr lupus mice;It can regulate the expression of lncRNA GAS5/miR-21-5p/Sprouty1 combination and the activation of ERK/CREB pathway in the kidney of MRL/lpr lupus mice.(3)Qihuang Jianpi Zishen Granules inhibited the activation of ERK/CREB pathway,inhibited the proliferation of mesangial cells and improved the damage of SLE kidney through the combination of lncRNA GAS5/miR-21-5p/Sprouty1. |
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