| Objective: to investigate the preventive and therapeutic mechanism of action on post-menopause osteoarthritis by supplementing the kidney and quickening the blood method through animal models experiment.Material and methods: 196 female SD rats were randomly divided into 6 groups, group A(48 rats, Received oral TCM), group B(48 rats, Received voltaren), group C(48 rats, model group), group D(30 rats, only undergoing A-PCL transection), group E(14 rats, normal blank group), group F(8 rats, ovariectomy group). Rats group A is divided in 3 groups, as group Al,group A2 and group A3, and tissue were harvested at three times. Every group was subdivided into 2 groups, as Al. 1, Al. 2, A2. 1, A2.2 , A3. 1 , A3. 2. At every tissue harvest, tibia bone histomorphometry was measured in the first group, with global pathology observation and immunohistochemistry of COL1, COL-2 measured in the second group. Group B and group C was designed as group A. Group D was divided into 3 groups, as group D1, group D2, group D3(10 rats each group). Every group was subdivided into 2 groups, as D1.1, D1.2, D2.1, D2. 2, D3.1, D3.2, (3 rats in the first group and 7 in the second group). The measurement of markers in both group was the same as in group A. Group E, used in the third tissue harvest, was randomly divided into two groups (7 rats each group), with the same measurement markers as group A. Group F, used in the third tissue harvest, was randomly divided into two groups (4 rats each group), with the same measurement markers as group A. Bone mineral density (BMD) was measured in the distal femur and serum estradiol levels measured in all rats. Bilateral ovariectomy, using Pernille surgical procedures, was done anesthetized by abdominal injection of chloral hydrate at 10% concentration in group A, group B and group C. Longitudinal skin and facial incisions were made over the lateral side of the right knee. The A-PCL was transected understerile conditions using Me Devitt surgical technique. After surgery, synovium, fasciae and skin were sutured. In group D, bilateral ovaries were pulled out and after partial resection of the fat, were laid back in the abdominal cavity. The next steps were the same as group A. Group E Received no surgical intervention. In group F, Bilateral ovariectomy was performed and ACL and PCL retained after incision of right knee skin. All the incision were administered with penicillin and all the rats received daily, intramuscular 40000 U penicillin for three days post surgery.The supplementing kidney and quickening the blood medicine consisted of semen, radix, astragali, and so on, at the concentration of 1.9926g/ml(supplied by the Affiliated Traumatology Hospital of GUTCM). Voltaren, at the concentration of 1.0875mg/ml, using Sodium Chloride as control.At one month and a half post surgery, post-menopause osteoarthritis animal model gained success. Group A were treated with supplementing the kidney and quickening the blood medicine oral once daily(6ml/kg/day); Group B were treated with voltaren oral once daily(9. 8mg/kg /day); Group B> C D^ E and F were treated with Sodium Chloride oral once daily(2ml Per rat).Results :Serum estrogen level: After induction of rat model, At weeks 2 post treatment with responding medicine, the serum estrogen levels of group Al, Bland Cl decreased compared with group Dl, with significant difference (P<0. 01) ;the serum levels of group Dl matched with group E, group Bl matched with group Cl, without significant difference (P>0. 05) ;These data suggested that ovariectomy resulted in decrease of serum estrogen levels in rats. At weeks 4 and 8 compared with at 2 weeks post intragastric administration, the serum estrogen levels in group E and D showed no significant difference (P >0. 05) ;the serum estrogen levels of group A3 were significantly higher than group A2, Al but lower than normal values, with significant difference(P<0. 05). The serum estrogen levels of group A increased significantly than group B, C, D, E, F, with significant difference (P<0. 05) ; the serum estrogen levels of group B, C, F decreased significantly than group D, E, with significant difference (P<0. 05) ; These data suggested that traditional Chinese medicine could improve estrogen levels, but voltaren and sodium chloride had no effects on estrogen levels.pathology changes:observation under naked eyes and ordinary microscope: tissue were harvested at weeks 2 post intragastric administration. In group A, cartilage surface were not smooth and surface layer chondrocytes disappeared, with formed pannus on cartilage surface, regularly arranged chondrocytes, appearance of chondrocyte cluster; group B exhibited unsmooth cartilage surface, in some cases showed niche in cartilage surface deep into reflecting layer, superficial chondrocytes disappearance, appearance of chondrocytes cluster in transitional zone, atrophy of chondrocytes nucleus in reflecting layer, slight cell cluster in calcified zone;group C exhibited unsmooth cartilage surface, surface thinning and erosion, in some areas showed villiform appearance. Tissue harvested at weeks 4 post intragastric administration: group A exhibited smooth cartilage surface, dissaperance of superfacial chondrocytes, lackness of pannus formation, regularly arranged chondrocytes, less chondrocytes cluster than weeks 8; group B exibited unsmooth cartilage surface, dissaperance of superfacial chondrocytes, appearance of chondrocytes cluster in transitional zone, atrophy of chondrocytes nucleus in reflecting layer, slight cell cluster in calcified zone; group C exhibited unsmooth cartilage surface, absence of superfacial chondrocytes, irregularly arranged chondrocytes, in some cases showed niche in cartilage surface deep into reflecting layer and calcified zone, tissue harvested at weeks 8 post intragastric administration: group A exhibited generally smooth cartilage surface, visible superficial chondrocytes, no pannus formation, regular choodrocytes eirrangement. Group B exhibited generally smooth cartilage surface, partial superficial chondrocytes existence, chondrocytes cluster in two samples, and fissure deep into transitional zone in one sample. Group C exhibited erosion-like change, absence of superficial chondrocytes, irregular chondrocytes arrangement, presence of cartilage fissure deep into reflecting zone and calcified zone. Changes in group D and F were the same as group C. Group E exhibited smooth cartilage surface, presence of superficial chondrocytes, regular cell arrangement.HHGS grading: 2 weeks post intragastric administration, the HGSS scores of group A and B failed to show statistical significance; the HGSS scores of group A and B were significantly difference compared to group C and D (P< 0. 01) . At 4 weeks post intragastric administration, the statistical analysisrepeated the 2 weeks' results. 8 weeks post intragastric administration, the HGSS scores of group A failed to show statistical significance compared with group B (P>0. 5) , but there were significant difference compared with 2,4 weeks post treatment; the HGSS scores of group A and B were significantly difference compared to group C, D and E (P<0. 01) . the HGSS scores of group A and B were significantly difference compared to group F (P<0. 05) ;These data showed that traditional Chinese medicine and voltaren had excellent therapeutic efficacy, but failed to recover the articular cartilage lesion to normal level in a short run. the HGSS scores of group C,D and c were significantly difference compared to group F (P<0. 01)Immunohistochemistry changes:The present study evaluated the OA cartilage immnohistochemistry staining using a grading system of slight, moderate or severe lesion compared with normal staining. At weeks 2 post intragastric administration, the tissue sample of group A, B, C and D exhibited severe lesion. 2 weeks post intragastric administration, group C and D continually exhibited severe lesion, and group A and B showed cartilage lesion between moderate and severe degree. 8 weeks post intragastric administration, group C and D continually exhibited severe lesion, and group A showed slight cartilage lesion, with group B exhibiting moderate cartilage damage, with group F exhibiting moderate cartilage ? damage, and group A is normal.Bone histomorphometry changes:Proximal left tibia cancellous bone histomorphometry changes in ovariectomized rats: in the first tissue harvest, %Tb. Ar, Tb. N> BFR/TV decrease and Tb. Sp increase in group Al, Bl, Cl compared to in group Dl, with significant difference. Static parameters of group al somehow improve compared with group Bl, Cl. %L. Piik BFR/BS, BFR/BV, Oc. N/mnu Oc. N/mm2 increased (P>0. 05) ; MAR decreased (P>0. 05) ;Suggesting that ovariectomized kidney vacuity animal model were induced, and with high turnover bone metabolism, osteoporosis developed in left knee joints gradually.In the second tissue harvest, %Tb. Ain Tb. 1^ BFR/TV decrease and Tb. Sp increase continually in group A2, B2, C2 compared to in group D2, with significant difference (P<0. 01) demonstrating that kidney vacuity resulted in further osteoporosis in left tibia of the rats. Static parameters of group a2 improve further compared with group B2, C2, suggesting that traditionalChinese medicine had effects on treatment of osteoporosis. %L.Puk BFR/BS, BFR/BV, Oc. N/mrru Oc. N/mm2 continually increased (P>0. 05) ; MAR continually decreased (P<0. 05) Suggesting that osteoporosis tended to developed further more.At the end time point, %L. Pnu BFR/BS, BFR/BV, BFR/TV continually increased in group A3 compared with group F, with significant difference (P<0. 05, 0.01) . %L. Pm , BFR/TV and MAR tended to increase (P>0. 05) ; 0c. N/mm and 0c. N/mm2 tended to decrease (P>0.05) . These data suggested that osteoporosis improved and traditional Chinese medicine had certain effects on increasing the number and cell viability of osteoblasts. %Tb.Ar, Tb. N> BFR/TV continually decreased in group B3 and C3 compared with group D3 and E, and Tb. Sp continually increased, with significant difference (P<0. 01) ; %L. Pm, BFR/BS, BFR/BV, Oc. N/mnu Oc. N/mm2 tended to continually increased (P>0. 05) ; MAR tended to continually decrease (P>0. 05) . There were no significant defference between group B3, C3 and group F. In group Dl, D2 and D3, parameters kept static changes, almost the same as group E.Comparison of static parameters of right tibia in 3 times of tissue harvest:%Tb. Ar, Tb. N increased significantly in group D compared with group A, B, respectively, and Tb. Sp decreased, with significant difference (P<0. 01); Tb. Th increased. At the end time point, the above three static parameters also showed statistically significant difference in the comparison of group D3 with group E. The stcitic parameters improved gradually during three times of tissue harvest in group A, B and C; changes in group A better than group B and C, with significant difference (P<0. 05) .These data demonstrated that traditional Chinese medicine had better efficacy on 0A than the other two groups. At the end time point, %Tb. Ar, Tb.N increased significantly in group A3,B3 and C3 compared with group F, and Tb. Sp decreased, with significant defference (P<0.01) . At the end time point, %Tb. Ar, Tb.N decreased significantly in group A3, B3 and C3 compared with group E, and Tb. Sp increased, with significant difference (P<0. 01) .These data showed that bone histomorphometry somewhat improved in group A3, B3 and C3 compared with in group F(single ovariectomy), but still didn' t reach the normal level compared with blank group.Comparison of dynamic parameters of right tibia in 3 times of tissue harvest:MAR of group D increased significantly compared with group E in three times of tissue harvest, with significant difference (P<0. 01) ;The values of other groups were lower than normal group, suggesting that there were significant proliferation of osteoblasts in the onset of osteoarthritis; relatively normal %L. Pm in each group suggested the number of osteoblasts showed no significant difference. BFR/BV of each group were significantly higher compared with group E in three times of tissue harvest, with significant defference (P<0. 01) ;Suggest ing that there was higher bone turnover rate in the onset of 0A. BFR/TV of group B and C showed statistically significant difference (P<0. 01) compared with group E; BFR/TV, %L. Pm ? MAR, BFR/BV of group A in three times of tissue harvest were higher than responding group B and C respectively, with increasing trend, suggesting that traditional Chinese medicine had certain effects on increasing the number and cell viability of osteoblasts. XL. Pm , MAR of group A3 showed statistically significant difference (P<0. 05) compared with group D3, suggesting that traditional Chinese medicine had better efficacy on OA than Western medicine group. Both bone formation and bone resorption parameters in group F were higher than in group D3, although without significant difference, these results suggested that there were some difference of bone turnover between single ovariectomy and single A-PCLT.Comparison of bilateral tibia histomorphometry of group A, B, C, D with group E, F%Tb. Ar, Tb. N> MAR of left limb in group A were lower than right limb in three times of tissue harvest, with Tb. Sp, Oc. N/mm2 higher than right limb. %Tb. Ar, Tb.N were lower compared with single A-PCLT(group D), but Tb.Sp were higher, with significant difference(P<0. 01). %Tb. Ar,Tb. N were higher compared with single ovariectomy(group F). The same trend existed in group B and group C. %Tb. Ar, Tb. NU 0c. N/mm2 of right limb in group D were lower than left limb in three times of tissue harvest, with Tb.Sp, MAR higher than right limb.Measurement of BMD:At weeks 2, 4 and 8 post administration with responding medication, BMDof distal metaphysis and shaft of right femur increase gradually in group A, Band E, but the difference between A2 and Al was not statistically significant(P>0. 05) . BMD data were analyzed between A3 and A2 with significance atP< 0.05 and between group B and group E without significance at P>0. 05. In...
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