| PART I Ameliorative effect of Drug pair of Radix Rehmanniae and Cornus officinalis、Its Containing serum、Its effective parts and Its active ingredientson AGEs-collagen cross-linking in vitroObjective:To observed the ameliorative effect of Drug pair of Radix Rehmanniae and Cornus officinalis. Its effective parts and Its active ingredients. Its Containing serum on cross-linking of AGEs with rat tail tendon collagen in 96-well plates. ELISA was used to determine the inhibitory of them.Methods:1. Preparation of AGEs modelBovine serum albumin BSA 50 mg/ml with 0.5 mol/L Glucose incubated together in Phosphate buffer for three months in vitro to prepare AGEs(AGEs-BSA). Preparation of sugar-free of bovine serum albumin at the same time.2. Preparation of AGEs with rat tail tendon collagen modelTake the dilution of AGEs into collagen-coated 96-well plates for four hours, the same concentration of BSA as a reference. To remove the uncoross-linked with PBST.3. Ameliorative effect of Drug pair of Radix Rehmanniae and Cornus officinalis. Its effective parts and Its active ingredients. Its Containing serum on AGEs-collagen cross-linking in vitro. Drugs added into cross-linked of AGEs and BSA, PBS added as a Non-inhibited control in the same way. The inhibitory rate of the drugs:(ODPBs-ODdrag)/ODPBS×100%(the value of OD used the average of the three).Result:1. Fluorescence scanning of AGEs(395/460nm):AGEs-BSA:34.4±1.1,0-BSA:8.2±0.4。 content of AGEs-modified protein for 4mg/ml.2. When the crude drug concentration is 0.03125g/L～0.5g/L, the percentage of inhibition on the AGEs-collagen cross-linking by Radix Rehmanniae、Cornus officinalis、the combine decoctions of Radix Rehmanniae and Cornus officinalis、the Separate decoctions of Radix Rehmanniae and Cornus officinalis for-3.2～1.1%,-6.1～-0.5%,-2.84～9.99%,-0.27～1.54%. 12 times the of Clinical amount of administration obtained Containing serum from 20% to 100%, The percentage of inhibition on the AGEs -collagen cross-linking by the Containing serum of Radix Rehmanniae、Cornus officinalis, the combine decoctions of Radix Rehmanniae and Cornus officinalis、the Separate decoctions of Radix Rehmanniae and Cornus officinalis for-3.2～10.4%、-4.7～15.6%、-3.6～19.9%,-4.1～13.7%.3. When the effective site concentration is 0.03125g/L～0.5g/L(meter of crude drug),the percentage of inhibition on the AGEs-collagen cross-linking by the iridoid terpene glycosides of Radix Rehmanniae(A)、oligosaccharide of Radix Rehmanniae(B)、poly saccharose of Radix Rehmanniae(C)、iridoid terpene glycosides of Cornus officinalis(D)、polysaccharose of Cornus officinalis(E)、triterpene acid of Cornus officinalis(F) for 1.7%～8.8%,-0.2%～7.9%,0.6%～ 7.3%,0.6%～6.8%,0.2%～4.9%,-1.2%～2.7%. the effective parts of Radix Rehmanniae and Cornus officinalis were composed through orthogonal design according to Lis (37). A1B1C1D2E2, A1B1C1D1E1F1 were better than the others.4. The active ingredient in 1 ×10-7～1×10-5 mol/L within, the percentage of inhibition on the AGEs-collagen cross-linking by the stachyose、Catalpol、Verbascoside of Radix Rehmanniae for-1～6.5%,-2.9～7.0%,-3.6～16.8%; The percentage of inhibition on the AGEs-collagen cross-linking by the Oleanolic acid、Ursolic acid、Loganin、Morroniside、Dishes glycosides of Swertia of Cornus officinalis for-7.8～5.2%、0.4～17.4%、-2.0～12.0%、-0.4～8.1%、-5.6-11.7%.Conclusion:The percentage of inhibition on the AGEs-collagen cross-linking by the combine decoctions of Radix Rehmanniae and Comus officinalis is better than the Separate decoctions; The percentage of inhibition on the AGEs-collagen cross-linking by Containing serum of the combine decoctions of Radix Rehmanniae and Comus officinalis is better than the Separate decoctions; the most obvious of the percentage of inhibition on the AGEs-collagen cross-linking by the effective parts of Radix Rehmanniae and Comus officinalis for Comus officinalis ursolic acid; and the effect on dose-dependent manner. So the combine decoctions of Radix Rehmanniae and Comus officinalis was the best.PART II Role and mechanism of the drug pair of Radix Rehmanniae and Cornus officinalis of preventing the damage of human umbilical vein endothelial cells cultured in AGEs mediumObjective:To study and research the role and mechanism of the drug pair of Radix Rehmanniae and Cornus officinalis of preventing the damage of human umbilical vein endothelial cells in AGEs medium.Methods:1. The establishment of HUVEC injury model in AGEs medium.human umbilical vein endothelial cells(HUVEC)were induced by different concentrations of AGEs(0、50,100,200,400 mg/L) and the cellular morphology was observed, at the same time we toke without AGEs group and glucose-free bovine serum albumin (BSA) group (concentration of 200mg/L) as the control. The cell proliferation was measured by CCK-8 method(450nm).2. The influence (proliferation rate) of the drug pair of Radix Rehmanniae and Cornus officinalis on the normal HUVEC10%FBS RPMI-1640 medium with 200mg/L AGEs and the drug pair of Radix Rehmanniae and Cornus officinalis(final concentration for 0.5g/T、0.250 g/L、0.125g/L.0.0625 g/L、0.03125 g/L) added to the cell plates, at the same time the 10%FBS RPMI-1640 medium as normal control. The cell proliferation was measured by MTT method(490nm).3. the role of the drug pair of Radix Rehmanniae and Cornus officinalis of preventing the damage of human umbilical vein endothelial cells in AGEs medium.10%FBS RPMI-1640 medium with 200mg/L AGEs and the drug pair of Radix Rehmanniae and Cornus officinalis(final concentration for 0.125g/L、0.0625 g/L、0.03125 g/L) added to the cell plates, at the same time the 10%FBS RPMI-1640 medium as normal control; Aminoguanidine as a positive control. The cell proliferation was measured by MTT method(490nm).4. the role and mechanism of the drug pair of Radix Rehmanniae and Cornus officinalis of preventing the damage of human umbilical vein endothelial cells in AGEs medium.10%FBS RPMI-1640 medium with 200mg/L AGEs and the drug pair of Radix Rehmanniae and Cornus officinalis(final concentration for 0.125g/L、0.0625 g/L、0.03125 g/L) added to the cell plates, at the same time the 10%FBS RPMI-1640 medium as normal control; Aminoguanidine as a positive control. The contents of SOD、MDA and LDH in HUVEC supernatant are measured to evaluate oxidative stress. The contents of NO、 vwF、TNF-α and IL-1β supernatant are measured to evaluate secretory function of the HUVEC.Result:1. The HUVEC 48h with different concentrations of AGEs (400,200,100,50 mg/L), the results show that with the concentration of AGEs increases, the inbibitional effect of HUVEC were significantly enhanced, AGEs (100,200,400 mg/L)were significant difference compared with the normal control group(P<0.05,0.01).200 mg/L AGEs stimulation HUVEC for 0,6,12,24,48h and found that cell proliferation was uninhibited When 6h,12h,(P> 0.05), cell inhibition was significantly increased after 24h by 200 mg/L AGEs(P<0.01).2. Radix Rehmanniae(0.125g/L,0.0625g/L) become a significant promoting role on the growth of HUVEC (P<0.01, P<0.05), On the contrary,0.5g/L and 0.25g/L inhibit. Cornus officinalis(0.125g/L-0.03125g/L) become a significant promoting role on the growth of HUVEC (P<0.01),0.5 g/L and 0.25g/L inhibit, the combine decoctions of Radix Rehmanniae and Cornus officinalis (0.5 g/L-0.03125g/L) become a significant promoting role on the growth of HUVEC,0.125g/L-0.03125g/L were significant (P<0.05, P<0.01); the Separate decoctions of Radix Rehmanniae and Cornus officinalis(0.125g/L-0.03125g/L) become a significant promoting role on the growth of HUVEC, in the 0.5g/L-0.25g/L concentration were inhibit.3. AGEs with HUVEC for 24 hours, compared with normal control group, AGEs reduce cell viability of HUVEC significantly (P<0.01); at the same time, different concentrations of the drug pair of Radix Rehmanniae and Cornus officinalis (0.125g/L,0.0625g/L,0.03125g/L) with HUVEC for 24 hours, Radix Rehmanniae group(0.125g/L,0.0625g/L) and Cornus officinalis group (0.125g/L,0.0625g/L) with AGEs induced HUVEC injury was significantly of Protective effect (P<0.05, P<0.01). the combine decoctions of Radix Rehmanniae and Cornus officinalis (0.0625g/L and 0.03125g/L) and the Separate decoctions of Radix Rehmanniae and Cornus officinalis (0.0625g/L and 0.03125g/L) with AGEs induced HUVEC injury was significantly of Protective effect (P<0.05, P<0.01). the combine decoctions of Radix Rehmanniae and Cornus officinalis were better than the other groups.4. AGEs with HUVEC for 24 hours, take the supernatant of HUVEC was measured of oxidative stress and cytokines:SOD, MDA, NO and LDH, vwF, TNF-a and IL-1β. compared with normal control group, the results showed that the supernatant of AGEs group was reduced the level of SOD, NO; increased the level of MDA, LDH (p<0.01). Point that HUVEC were damage, antioxidant capacity were reduced. TNF-a, IL-1β,vwF factor secretion were increased, Point that the secretion dysfunction of the HUVEC, inflammation were increased, the drug pair of Radix Rehmanniae and Cornus officinalis could enhance the activities of SOD, NO; to reduce the level of MDA, LDH; to decrease the cytokine secretion of vwF, TNF-a and IL-1β; and compared with AGEs group were significant differences (p<0.05,0.01). Among them, the combine decoctions of Radix Rehmanniae and Cornus officinalis were better than the other groups.Conclusion:Drug pair of Radix Rehmanniae and Cornus officinalis, Radix Rehmanniae, Cornus officinalis all preventing the damage of human umbilical vein endothelial cells in AGEs medium, the combine decoctions of Radix Rehmanniae and Cornus officinalis was the best, its protective mechanism may be through inhibition of oxidative stress and restore some the dynamic balance of NO, reduce vwF content, reducing reducing the secretion of IL-1β and TNF-α protein to regulate HUVEC secretory function to protect endothelial cells. |
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