Study On The Renoprotective Effect Of Spontaneously Hypertensive Rats And Underlying Mechanism Of Meicha Total Flavonoids And Meicha Protein

Object: In this study herein,we firstly investigated the effect of Meicha total flavonoids and Meicha protein through the combination treatment of spontaneously hypertensive rat(SHR)on Hypertension kidney damage.By virtue of investigating TLR4 signal pathway and renin angiotensin system explored the underlying mechanism in vivo.Furthermore,we explored the specific mechanism of the combination treatment of Meicha total flavonoids and Meicha protein on TLR4 signal pathway in vitro.Methods: 1.Effect of the combination treatment of Meicha total flavonoids and Meicha protein on the kidney of SHR 40 healthy SHR rats were randomly divided into four groups of ten: the SHR group,Meicha total flavonoids group,Meicha protein group,combination group.Adaptive feeding rats and measuring blood pressure for 3W,the SHR group were orally given volume of distilled water,gavage capacity of 10 m L.kg-1 body weight,the Meicha total flavonoids group dose was 180 mg.kg-1,Meicha protein group dose was 70 mg.kg-1,the combination group dose was total flavonoids Meicha 180 mg.kg-1 and Meicha protein mg.kg-1.The rats orally administered twice daily gavage seven weeks,blood pressure was measured weekly and record each group.Before the last administration 12 h,only water is available.After the last administration,renal tissue should be stripped off rapidly.Dry them off by filter paper and weigh precisely,afterward calculate organ index(organ index = organ weight / body weight).Take part of the tissue with 4% paraformaldehyde for HE staining.Observe the morphology under an optical microscope.2.The combination of Meicha total flavonoids and Meich protein on the underlying mechanism of the renoprotective effect of SHR Grouping and method of administration are the same with 1.After the last administration,sacrificed the SHR.Renal tissue should be stripped off rapidly and stored at-80 ℃.They were as a real-time PCR test samples to detect the m RNA expression of TLR4,NF-κB,IL-6,TNF-α,ACE,AngⅡand CYP11B2.In addition,the protein expression of TLR4 and NF-κB in renal tissue of SHR in each group were measured.3.Research on the effect of the combination of Meicha total flavonoids and Meich protein on the TLR4 signal pathway of renal tubular epithelia cells(NRK-52E)NRK-52 E were maintained in DMEM medium supplemented with 5% fetal bovine serum.NRK-52 E were pre-incubated in either the presence or the absence of AngⅡ(c=10-6mol/l)for 24 h.Then the NRK-52 E which had been pre-incubated in AngⅡwould be respectively stimulated by the Meicha total flavonoids,the Meicha protein and the combination of the two for 24 h.Eventually,the m RNA expression of TLR4,NF-κB,IL-6,TNF-α were measured.The protein expression of TLR4 and NF-κB in NRK-52 E were measured as well.Results: 1.The change of body weight,systolic blood pressure and the renal index in each group: until the end of the experiment,body weight did not differ among the four groups.Compared to the SHR group,the Meicha total flavonoids group,the Meicha protein group,the combination group can significantly reduce blood pressure in SHR.The impact of organ coefficient Meicha total flavonoids and Meicha protein in SHR was not significant.2..Renal pathological changes: The typical pathological characteristics and infiltrative inflammatory cells in hypertensive renal injury were demonstrated in the kidney of SHR in the SHR group,and the hypertensive renal injury was ameliorated after treatment with the combination of Meicha total flavonoids and Meicha protein.3.The m RNA expressions of ACE,Ang Ⅱ and CYP11B2 in SHR: by comparison with the SHR group,the m RNA expressions of Ang Ⅱ was significantly decreased(P<0.05),and the m RNA expressions of ACE and CYP11B2 was significantly decreased in Meicha total flavonoids group(P<0.01);The m RNA expressions of AngⅡ,ACE and CYP11B2 were significantly decreased in the combination group(P<0.01);There was no difference between the SHR group and the Meicha protein group(P>0.05).Moreover,compared the combination group with the Meicha total flavonoids group,the m RNA expressions of ACE,AngⅡand CYP11B2 were significantly decreased(P<0.05).4.Compared with the SHR group,the m RNA expressions of TLR4,NF-κB,IL-6 and TNF-α were significantly decreased both in the combination group and the Meicha total flavonoids group(P<0.01);There was no difference between the SHR group and the Meicha protein group(P>0.05).Compared with the SHR group,the protein expressions of TLR4 and NF-κB in the three of all groups were significantly decreased(P<0.01).Moreover,Compared the combination group with Meicha protein group,the protein expressions of TLR4 and NF-κB in the combination group were significantly decreased(P<0.01).5.The m RNA expressions of TLR4,NF-κB,IL-6 and TNF-α in AngⅡ-induced NRK-52E:by comparison to the control group,the m RNA expressions of TLR4,NF-κB,IL-6 and TNF-α in Ang Ⅱ group were significantly increased(P<0.01).Compared with the AngⅡgroup,the m RNA expressions of TLR4,NF-κB,IL-6 and TNF-α in the Meicha total flavonoids group were significantly decreased(P<0.05);The m RNA expressions of TLR4,NF-κB,IL-6 and TNF-α were significantly decreased in the both combination group and the Meicha protein group(P<0.01).Moreover,Compared combination group with Meicha total flavonoids group,the m RNA expressions of TLR4,NF-κB and IL-6 in the combination group were significantly decreased(P<0.01);The m RNA expressions of TNF-α was significantly decreased(P<0.05).6.The protein expressions of TLR4 and NF-κB in AngⅡ-induced NRK-52 E cells:compared with the control group,the protein expressions of TLR4 and NF-κB in AngⅡgroup were significantly increased(P<0.01).In comparison to the Ang Ⅱ group,the protein expressions of TLR4 were significantly decreased in Meicha total flavonoids group(P<0.05),the protein expressions of NF-κB were significantly decreased in Meicha total flavonoids group(P<0.01);The protein expressions of TLR4 and NF-κB were significantly decreased in both the combination group and the Meicha protein group(P<0.01).Moreover,Compared the combination group with two of the singel group,the protein expressions of TLR4 and NF-κB in the combination group were significantly decreased(P<0.01).The results indicated that combination of Meicha total flavonoids and Meicha protein is superior to single alone in the renal protection.Conclusion: 1.The combination of Meicha total flavonoids and Meicha protein can protect the kidney of SHR from hypertensive injury.2.The main mechanism of the synergistic renal protection effect contributed to the combination of Meicha total flavonoids and Meicha protein was that through cooperative inhibition of the m RNA expression of ACE,AngⅡand CYP11B2 in the kidney of SHR and inhibition of TLR4 signal pathway.