| ObjectiveTo observe the effects of the combination of classic formula of “Shanghan Lun”,Didang decoction and Xiao Xianxiong decoction,namely Didang Xianxiong decoction which has the effect of resolving phlegm,removing blood stasis and dredging collaterals on the renal microangiopathy of diabetes mellitus(DM)rats.Therefore,the RAGE/NOX4/NF-κB pathway,which is mainly associated with AGEs,was used as a target to investigate the mechanism of the effect of Didang Xianxiong decoction on the renal microangiopathy of DM,using the indicators related to the microangiopathy of DN.This study provides a scientific and theoretical grounds for the clinical application of classic decoction in the prevention and treatment of DN.MethodsOne hundred SPF-grade SD male rats with a body mass of 200±20 g were selected for the experiment.After 1 week of acclimatization feeding,15 rats were taken according to the random number table method as the bank control group(group N),and the rest of the 85 rats were used for DM renal lesion rat model establishment.The rats were fasted without water for 24 h before modeling.Firstly,a diabetes mellitus(DM)model was established by streptozotocin solution(STZ)with a single intraperitoneal injection in 55 mg/kg.The successful DM rats were fed for 4 weeks,during which the rats that did not meet the blood glucose standard and those that died of infection were removed,resulting in 60 rats that fit the DM renal lesion model.The DM with renal lesion rats successfully moulded were divided into 5 groups of 12 rats each according to the random number table method,namely model group(group M),Didang Xianxiong decoction group(group R),Xiao Xianxiong decoction group(group X),Didang decoction group(group D)and the ALT-711 group(group P).Then the rats were gavaged once/d daily at a fixed time of 9 a.m.for 8 weeks with doses of 8.1 g/kg,4.05 g/kg,4.05 g/kg in group R,D and X respectively.Group P was given ALT-711 solution at a dose of 3 mg/kg and a gavage volume of 10 m L/kg,while group N and M were gabaged with equivalent volume of distilled water.The rats were fasted without water for 12 h after the final gavage and taken samples under anesthesia.(1)The changes in the general condition of rats were observed and recorded during the experimental period.(2)The effect of Didang Xianxiong decoction on renal microangiopathy in rats with DM:(1)Detection of VCAM-1 and VEGF m RNA expression in kidney tissues by RTq PCR;(2)Detection of VCAM-1and VEGF protein expression in kidney tissues by Western blot.(3)The effect of Didang Xianxiong decoction on indicators related to the RAGE/NOX4/NF-κB pathway:(1)Detection of RAGE,NOX4,p-NF-κB protein expression in kidney tissues by immunohistochemistry;(2)Detection of RAGE,NOX4 and NF-κB m RNA expression in kidney tissues by RT-q PCR;(3)Renal tissue RAGE,NOX4,NF-κB and p-NF-κB protein expression were detected using Western blot.Results(1)During the experiment,it was observed that DM rats with successful modeling has a slightly yellowish coat,lost their glossiness,increased excretion,especially in urine,increased water consumption and depression,which was more obvious at the end of the experiment.Although each drug intervention group improved compared to the M group,it was still not as good as the N group.(2)Results of the effect of Didang Xianxiong decoction on renal microangiopathy in rats with DM:(1)Expression of VCAM-1 and VEGF m RNA in kidney tissues:compared with group N,the expression levels of VCAM-1 and VEGF m RNA were upregulated in kidney tissues of group M(p<0.01).After 8 weeks of pharmacological intervention,the above m RNA expression levels were significantly downregulated in all intervention groups in comparison to the M group(p<0.01).In comparison between the treatment groups,the down-regulation of VCAM-1 and VEGF m RNA expression in X and D groups was less significant than that in group R(p<0.01),while the downregulation of the above m RNA expression levels in group P was comparable to that in group R.(2)VCAM-1 and VEGF protein expression in kidney tissues: VCAM-1 and VEGF protein expression levels were significantly higher in the M group compared to the N group(p<0.01).The expression level of VCAM-1 protein in all intervention groups and VEGF protein in R and P groups were significantly lower than those in the M group(p<0.01),however,the difference between the VEGF expression level in the X and D groups and the M group was not statistically significant.In comparison among the intervention groups,the expression levels of VCAM-1 and VEGF in renal tissues of groups X and D were not as low as those of group R(p<0.01),while the expression levels of these proteins in group P and R were comparable.(3)Results of the effect of Didang Xianxiong decoction on indicators related to RAGE/NOX4/NF-κB pathway:(1)Detection of RAGE,NOX4,p-NF-κB protein expression in kidney tissues by immunohistochemistry: the results showed that the cytosol,cytoplasm and nucleus of the intrinsic cells in the kidney were covered with brown substances corresponding to each protein expression site in the M group,and the mean optical density was higher than N group(p<0.01).There was a significant reduction in brown areas in group R compared to group M,with a scattered distribution(p<0.01),and there were still small areas of brown material in group X,D and P,with a banded distribution,but less brown area than in group M(p<0.01).Comparison between the intervention groups revealed that the mean optical density of RAGE,NOX4 and p-NF-κB was lower in the R group than in the X,D and P groups(p<0.01).(2)Expression of RAGE,NOX4 and NF-κB m RNA in kidney tissues: by comparison to the N group,all the above m RNA expression levels were up-regulated in the M group(p<0.01).After 8 weeks treatment,the m RNA expression levels of RAGE and NOX4 in each intervention group and NF-κB in R,D and P groups were significantly downregulated by comparison to the M group(p<0.01),but the downregulation of NF-κB m RNA in X group was not significant.In comparison between the intervention groups,the down-regulation of RAGE,NOX4 and NF-κB m RNA expression levels in the X and D groups was less significant than that in R group(p<0.01),while the down-regulation of the above m RNA expression levels in the P group was comparable to that in the R group.(3)Expression of RAGE,NOX4,NF-κB and p-NF-κB protein in kidney tissues: RAGE,NOX4 and p-NF-κB protein expression levels were signigicantly higher in the M group compared to the N group(p<0.01).After 8 weeks of pharmacological intervention,the expression levels of RAGE and p-NF-κB protein in all intervention groups and the NOX4 protein in the R and P groups were significantly lower than those in M group(p<0.01).However,the differences in NOX4 protein expression levels between groups X and D and group M were not statistically significant.The expression levels of RAGE,NOX4 and p-NF-κB in the kidney tissues of the X and D groups were not as low as those of the R group(p<0.01),and the expression levels of these proteins in the P and R groups were comparable.NF-κB protein expression was not significantly different in all groups.Conclusion(1)The general condition of the rats with DM was improved by the effect of the Didang Xianxiong decoction.(2)The RAGE/NOX4/NF-κB pathway can be effectively inhibited,thus reducing the expression of VCAM-1 and VEGF,which can interfere with the renal microangiopathy of DM rats,and may be one of the mechanisms of the action of Didang Xianxiong decoction.(3)The combination of the classic formula Didang Xianxiong decoction has the best effect on the prevention and treatment of renal microangiopathy in rats with DM,which is significantly better than Didang decotion or Xiao Xianxiong decoction,which are used alone to remove blood stasis and dredge collaterals and resolve phlegm. |
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