| Objective:Extract and isolate total flavonoids of Desmodium styracifolium (TFDS) from Desmodium styraci folium, take L-hydroxyproline inducing rat renal calcium oxalate calculus model. Observe the TFDS effect on stones formation and rat biochemical indexes related to stones formation to determine curative effect. To explain the underlying mechanisms of antiurolithic by measuring indicator of oxidative stress and inflammation-related protein expressionMethods:Separation and purification of Desmodium styracifolium (TFDS) by macroporous resin, by LC-MS/MS technology for quality control. Take L hydroxyproline inducing rat renal calcium oxalate calculus model. Sprague-Dawley rats weighing 180-220 g were divided with matched body weights into five groups of eight animals each:group Ⅰ, control group, group Ⅱ serving as TFDS alone, group Ⅲ, serving as the stone group, received stone-inducing treatment, for up to 28 days, fed 5%L-hydroxyproline in their food, Treated groups Ⅳ, Ⅴreceived different dose of TFDS(100mg/kg/d) and (400mg/kg/d) respectively and simultaneously received stone-inducing treatment similar to group Ⅲ, At the end of 28 days of treatment, the animals were housed individually in metabolic cages. Collecting morning urine and blood for the crystal-luria study, part of each urine sample was acidifi ed to pH 2 with 5 m HC1. Both acidifi ed and non-acidifi ed urine samples were then centrifuged at 1500 × g for 10 min to remove debris and supernatants were stored at -20℃ until analysed. Acidified urine samples were tested for oxalate (Ox), calcium (Ca2+) and magnesium (Mg2+) and so on, while non-acidifi ed urine samples were used to determine the contents of citrate, uric acid (UA) the blood were tested Serum creatinine and blood urea nitrogen concentration. The animals were killed and both the kidneys were excised, rinsed in ice cold physiological saline and weighed. The right kidney was fi xed in 4% Paraformaldehyde and stained with haematoxylin and eosin (H & E), Calcium salt dyeing and Polarizer detection. then the determination of OPN, MCP-1, TGF-β and so on. The left kidney was worked into a 10% homogenate in ice cold PBS (50 mm, pH 7.4), centrifuged at 1500 × g, and the supernatant was used to assess malondialdehyde (MDA) and protein carbonyl contents, reduced glutathione and activities of various antioxidant enzymes.Results:TFDS Purity is 68.34% isolated from Desmodium styracifolium. LC-MS/MS preliminary analysis confirmed that contains isoorientin and isovitexin flavonoid glycosides, such as monomer composition. The renal calcium oxalate calculus model is successful.(1) General index and biochemical index:The weight of all rats was increased but the stone group weight gain is the least. (P<0.05) The food intake of stone group was decreased and increased after treated with TFDS (100mg/kg/d). (P<0.05). Kidney weight, drinking water and urine of stone group were significantly higher than the control group and TFDS group (P< 0.05). TFDS (400mg/kg/d) group kidney weight is lighter than stone group. Urine PH of stone group and TFDS (100mg/kg/d)group is significantly reduced (P< 0.05), but the TFDS (400mg/kg/d) group compared with stone group is rised obviously. A significant raised in 24 h urine oxalic acid of stone group compared control group (P<0.05), and decreased obviously after treated with TFDS (P<0.01), and the TFDS (400mg/kg/d) group is better with almost control group level. Urinary calcium (Ca2+), magnesium (Mg2+) and citrate of TFDS (100mg/kg/d) group was increased significantly (P< 0.05). Compared with control group and TFDS alone group, the urine protein was decrease (P< 0.05), however, it’s lower than the stone group(P<0.05). Serum creatinine and blood urea nitrogen in stone group was raised apparently (P< 0.05)and indicated the Kinney injure. TFDS (400mg/kg/d) and TFDS (100mg/kg/d) can reduce serum creatinine level but the two group had no significant difference (P>0.05). Urinary crystals:Control group and TFDS alone group had no urinary crystals in a field of 400 ×, but the stone group urine had a lot of hexahedral into envelope samples of calcium oxalate crystal and the TFDS treated group is less than the stone group (HLP+LTFDS, HLP+HTFDS vs. HLP:2.00±4.00, 0.83±1.33 vs.7.50±3.78, P<0.001)。(2) Renal pathology:From the Kinney H & E staining and calcium salt We can find that the renal of control and TFDS alone group were almost Normal, but we can find that the many intra-tubular birefringent crystalline deposits were seen under polarized light in all regions of the kidneys (cortex, medulla and papilla) of all animals in stone group and the The renal tubules were markedly dilated in the entire kidneys of these rats, what’s more the exfoliative cells and inflammatory cell infiltration, These changes facilitate CaOx crystal adherence and retention in renal tubules. The similar changes also occurred in the TFDS treated group, but lighter than the stone group, especially the TFDS (400mg/kg/d) group is more effectively. Calcium oxalate crystal grade: The stone group was (4.67±0.52) is much higher than other group (P<0.01), but it’s decreased after treated with TFDS (400mg/kg/d) and TFDS (100mg/kg/d) (P<0.01) and the calcium oxalate crystal grade between TFDS (400mg/kg/d) (1.67±0.52)and TFDS (100mg/kg/d) (2.17±0.75)had no significant difference. Kidney tissues immunohistochemistry showed that the protein expression of MCP-1、OPN and TGF-β in stone group is significantly enhanced (P<0.05), and the TFDS treated group compared with stone group was decreased but the TFDS (100mg/kg/d) compared stone group has no significant difference. Oxidative stress indicators:The stone-inducing treatment enhanced MDA content (P<0.01),and decreased the CAT, GSH-Px level of kidneys (respectively P<0.01,P<0.05), and the TFDS treatment induced the decrease of MDA and increase of GSH-Px (P<0.01), and TFDS (400mg/kg/d) and TFDS (100mg/kg/d) has no difference (P>0.05), in addition the TFDS (400mg/kg/d) can induce CAT increased (P<0.05).Conclusions:Macroporous resin can be used for purification and enrichment oftotal flavonoids from D. styracifolium, which has the characteristics of easy operation, low cost, highlight purification effect. The model established by L-hydroxyproline is successful and is worth promoting. TFDS has the effect of inhibit the formation of caicium stones in rats:1) TFDS treatments can reduce the 24 h urine oxalic acid concentration, increased urinary calcium and urinary magnesium and urinary citrate and antioxidant then inhibit the renal calcium oxalate stone formation in rats; 2) TFDS can reduce the generation of ROS and MDA and decrease the consumption of antioxidant enzymes to resist Cell oxidative stress injury;3) another possible mechanism is TFDS can down-regulation the expression of MCP-1, TGF-β and OPN in the renal. |
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