Danshen (Salvia Miltiorrhiza) Injection Suppresses Kidney Injury Induced By Iron Overload In Mice

Objectives: Iron plays an important role in many metabolic andbiological processes. However, excessive iron can accumulate in a widevariety of tissues, including the liver, heart and kidney, leading to oxidativeinjury. Currently, iron chelators are widely used to treat iron overload diseases.Despite the therapeutic effectiveness of these drugs, some disadvantages exist,for example, hepatic dysfunction, allergy, agranulocytosis and poor patientcompliance. Therefore, development of new treatments for iron overloaddiseases would be highly beneficial. The commonly used Chinese herbalmedicine Salvia Miltiorrhiza (Danshen) can inhibit lipid peroxidation andscavenge oxygen free radicals, so we hypothesized that it may be a potentialmedicine for kidney injury induced by iron overload. The aim of the presentstudy is to evaluate the effects of treatment with Danshen injection on ironoverload-induced kidney damage in mice and the potential mechanisms.Methods:75male Kunming mice were divided into the following fivegroups of15animals per group: control, iron overload, deferoxamine (DFO),low-dose Danshen (L-Danshen) and high-dose Danshen (H-Danshen) groups.Mice of the latter four groups were intraperitoneally (i.p.) injected with asingle dose of iron dextran50mg/kg/day, and the control group received an i.p.injection with isovolumic saline. Mice of the L-Danshen and H-Danshengroups were given i.p. injections of Danshen (3and6g/kg/day, respectively)at4h before the administration of iron dextran on the same day, while mice ofthe control group and iron overload group received isovolumic saline by thesame route. Mice in the DFO group were likewise injected i.p. at the dose of100mg/kg/day at4h before the administration of iron dextran on the sameday.On the14th day, the final body weight of each animal was recorded, and animals were anesthetized with sodium pentobarbital. Blood from each animalwas collected, and the serum was separated for further analysis. The levels ofblood urea nitrogen (BUN) and creatinine (CR) in serum were estimated byspectrophotometer using commercially available kits. The kidney was quicklyexcised and washed in ice-cold normal saline and frozen in liquid nitrogen.Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL) staining was performed using an In Situ Cell Death Detection Kitaccording to the manufacturer’s protocol. Total iron levels in kidney tissuewere measured by flame atomic absorption spectroscopy. The activities ofsuperoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) as well asthe content of malondialdehyde (MDA) in kidney tissues were estimated byspectrophotometer using commercially available kits. Kidney tissues werefixed with4%buffered paraformaldehyde, hydrated in ascending grades ofethanol, cleared in xylene and embedded in paraffin. Tissue sections wereprepared for Prussian blue to observe iron deposition. And other kidneysections were prepared for hematoxylin and eosin (H&E), periodic acid–Schiff(PAS) and Masson’s trichrome staining, to perform histological studies.Components of Danshen injection were analyzed by High-performanceLiquid Chromatography (HPLC).Results:1Contents analysis of Danshen injection by HPLCThe results determined that the actual concentrations of the majorcomponents of Danshan injection,3,4-dihydroxyphenyl lactic acid(Danshensu), protocatechuic aldehyde and salvianolic acid B, to be2.15,0.44and1.01mg/ml, respectively.2Effects of Danshen Injection on Body WeightIron overload caused a significant decrease in weight gain, comparedwith the control group (P <0.01). Meanwhile, Danshen and DFO treatmentmarkedly enhanced the weight gain, compared with the iron overload group (P<0.01, P <0.01and P <0.05, respectively).3Effects of Danshen Injection on Iron Deposition in Kidneys Observed by Prussian Blue StainingLarge numbers of blue-stained iron deposits were seen in the glomeruli ofkidney sections in the iron overload group, compared with the control group.Meanwhile, levels of blue stained iron deposits in the DFO and Danshengroups were significantly reduced compared with the iron overload group.4Effects of Danshen Injection on Morphological Changes in KidneysThe kidney sections of the control group appeared healthy with normaltissue structures. By contrast, the kidney sections of iron-overloaded micedisplayed marked histological changes. The scores for glomerular andtubulointerstitial lesions showed an approximately21-fold increase in thelesion areas compared with those in the control group (P <0.01). Aftertreatments of iron overload mice with low or high doses of Danshen injection,the lesion areas were decreased approximately by40%and50%, respectively,compared to the iron overload group (P <0.01).5Effects of Danshen Injection on Iron Level in Kidney HomogenateIron dextran resulted in a dramatic elevation of total iron in kidneysections. Treatment with Danshen injection significantly decreased the level ofiron (P <0.01).6Effects of Danshen Injection on Levels of BUN and CR in SerumCompared with control mice, BUN and CR levels were notably elevatedin the iron-overloaded animals (P <0.01); however, these levels were clearlylower in the Danshen and DFO groups, compared with the iron overload group(P <0.01).7Effects of Danshen Injection on SOD, GSH-Px and MDA in KidneyHomogenateCompared to those of the control group, SOD and GSH-Px activitieswere dramatically decreased in the iron overload group (P <0.01), whileDanshen and DFO treated mice showed only a slight decrease in theseactivities (P <0.01). Furthermore, the concentration of MDA was significantlyenhanced in the iron overload group compared with the control group (P <0.01), while Danshen and DFO treatment markedly inhibited the excess iron-mediated enhancement of MDA levels (P <0.01).8Effects of Danshen Injection on Apoptosis in KidneysApoptosis in tubular cells remained at a consistently low level in thecontrol group, while TUNEL-positive tubular cells were significantlyincreased in the iron overload group (P <0.01). Danshen and DFO treatmentdramatically decreased the extent of this apoptosis compared with the ironoverload group (P <0.01).Conclusions: Our results indicated that Danshen injection exertedsignificant renal protective effects in iron-overloaded mice, which wereclosely associated with the decrease of iron deposition and suppression of lipidperoxidation and apoptosis in the kidney.