Effects Of Extracts From Prunella Vulgaris L. On Urinary Stone Formation And Osteopontin Expression In Rats Urolithiasis Model With Oxalate Calcium

Calcium nephrolithiasis is the most common form of renal stone disease,with calcium oxalate(CaOx)being the predominant constituent of renal stones.Modern medicine consider that CaOx crystal nucleation, growth,aggregation,and adhesion to renal cells plays a important role during renal stones formation and retention.Current in vitro evidence implicates osteopontin(OPN)as one of several macromolecular inhibitors of urinary crystallization with potentially important actions at several stages of CaOx crystal formation and retention.Prunella vulgaris L is a traditional chinese herbal used to treat various diseases for hundreds of years.Chinese herbal had been also used for the cure and the prevention of urinary calculi for many years,but the effect and the mechanism of this use of chinese herbal medicine are unclear.In the study we examined the inhibitory effect of the Chinese herbal medicine Prunella vulgaris L on the formation of CaOx crystral in vitro and CaOx renal stones induced by ethylene glycol(EG)and ammonium chloride in rats in vivo.We also investigated the e.ect of Prunella vulgaris L on osteopontin(OPN)expression.Objective:To study the inhibitory effect of Prunella vulgaris on urinary oxalate calcium(CaOx)stone formation in vitro and to investigate the effects the extracts from Prunella vulgaris on urinary calcium oxalate stone formation and osteopont in(OPN)expression in urolithiasis model rats,and to explore the mechanism of Prunella vulgaris on the prevention of the formation from urinary calculi.Methods:The aqueous extract from Prunella vulgaris(Aq-P.V) (group C₁and C₂),50%methanolic extract from Prunella vulgaris (Me-P.V)(group D₁and D₂),and 100%Me-P.V(group E₁and E₂)were prepared.The effects of several extracts whose concentration were at 10 and 20 mg·ml^-1,respectively,on inhibiting CaOx crystal growth (inhibiting index,I.I)were determined by a seeded crystallization technique in vitro.Forty adult male wistar strain rats were randomly divided into five groups(N=8 each),group normal control(A),group stone formation(B), Group TAq-ect(C),group 50%TMe-ext(D),group 100%TMe-ext(E). The model rats with renal calcium oxalate stone formation were induced by intragastric administration ethylene glycol and ammonium chloride. After 4 weeks,the concentration of serum BUN,Cr,Ca²⁺,P,the 24h urinary excretion Ca²⁺,Oxalate(Ox),Mg²⁺and the content of renal tissue Ca²⁺,Mg²⁺were detected.All kidneys were removed and examined microscopically for possible CaOx crystals deposit and tubular dilatation in the kidney.The expression of OPN mRNA in the rats kidney were detected by in situ hybridization and RT-polymerase chain reaction(RT-PCR) techniques.Immunohistochemistry and western blotting methods were used to assessed the protein expression of OPN in rat renal tissue of every groups.Results:The in vitro experiment showed that I.I in group C₁,C₂, D₁,D₂,E₁,E₂ was 50.8±5.6%,79.6±4.5%,45.6±6.5%,77.2±5.9%, 30.6±4.6%and 68.5±5.8%,respectively.The Aq-P.V had the strongest inhibition with I.I of 79.6±4.5%in high concentration,and I.I of group C and D were more than group E,but there was no significant difference in all groups in low concentration.The serum concentration of Ca²⁺and P showed no significant difference in all rats.24h urinary excretion Ca²⁺,Ox and Mg²⁺showed no significant difference compared each group of administered extract from Prunella vulgaris with group B.The content of renal tissue Ca²⁺was significantly lower in group A,C,D compared with group B(P＜0.05), the content of renal Mg²⁺showed no significant difference in all groups. A large number of CaOx crystals and significant tubular dilatation were observed in group B.CaOx crystals and tubular dilatation in group C were significantly difference compared with group B,only a few CaOx crystals and few tubular dilatation were observed in group D,group E were analogous with group B.von Kossa's method clearly demonstrated the presence of calcium oxalate deposits in the kidney of the stone group and Prunella vulgaris group.No deposits were detected in the control rats.Quantitative analysis of the density of calcium deposits showed a significantly lower density of deposits in the Prunella vulgaris group compared to the stone group.In situ hybridization demonstrated the expression of OPN mRNA in both the distal and proximal convoluted tubules,the loop of Henle,and collecting ducts(mainly the medullary thick ascending limb of the loop of Henle), the glomeruli were negative.In kidney from the normal control group, OPN mRNA was detected in a small proportion of the loops of Henle in the renal medulla.The kidney from the stone and 50%TMe-ext group showed overexpression of OPN mRNA;the increase in the 50%TMe-ext group was relatively weak compared to the stone group.The expression level of OPN mRNA using RT-PCR was correspond to In situ hybridization.The OPN protein expression was observed in the kidney of all three groups rats.The OPN protein were found in both the distal and proximal convoluted tubules,the loop of Henle,and collecting ducts(mainly the medullary thick ascending limb of the loop of Henle), whereas no staining for the protein was detected in glomeruli in the renal cortex.In rats of normal control group,only a weak OPN staining was noted.The expression was enhanced in the stone group.50%TMe-ext-treated rats tended to have lower OPN protein expression than the stone group.It was similar to Immunohistochemistry staining that the expression of OPN protein detected by western blotting. Conclusion:The extract from Prunella vulgaris can significantly inhibited urinary CaOx stone formation in vitro,the inhibitory effect of Aq-P.V and 50%Me-P.V were better than 100%Me-P.V.Animal experimental showed that 50%Me-P.V can more significantly decrease the content of the renal tissue Ca²⁺and tubular dilatation than Aq-P.V did in the in vivo experiment.The extracts from 50%Me-P.V Prunella vulgaris can inhibit the OPN expression and deposit of calcium oxalate in renal tissue so as to block the formation of renal calcium oxalate stone in rats potently.