| Objective First, to conduct clinical observation and analysis on the effects of Fallopia multiflora decoction for patients with DM and concomitant osteoporosis. Second, to build DM rat models and observe the dynamic changes in the bones of DM rats from four aspects including morphology, pathology, ultrastructural pathology and molecular biology, and understand the effects of Fallopia multiflora on the bone morphology and components of DM rat models.Methods Experiment one: Randomly selected 50 patients with DOP(diabetic osteoporosis) in clinical practice and divide into Group A(25 patients, foundation treatment group) and Group B(25 patients, Fallopia multiflora treatment group). Basic treatment in combination with insulin for control of blood glucose were provided in Group A, and additional Fallopia multiflora for oral treatment besides the basic treatments and insulin for control of blood glucose were provided in Group B. The changes of serum calcium, serum phosphorus, alkaline phosphatase, osteocalcin, bone mineral density and clinical VAS, QOL, SF-MPQ and numbness score respectively in Week 0,Week 4, Week 8 and Week 12 were observed.Experiment two: 100 rats were selected and were divided: group A is blank control group(n=20); the rest of rats were prepared into diabetic and osteoporosis rat models by being treated with STZ for 8 weeks, then 60 successful models from those rats were selected and were randomly divided into 3 groups. : 20 rats in DM group(Group B), 20 rats in insulin treatment group(Group C) and 20 rats in Fallopia multiflora group(Group D). Appropriate interventions were performed for the experimental animals in each group and the animals were sacrificed respectively in Week 0,Week 8, Week 12 and Week 16. Weight, Blood sugar, Glycosylated hemoglobin, Blood fat,BMD(bone mineral density), serum phosphorus, Serum calcium, alkaline phosphatase, and osteocalcin were detected,; and in batches executed them in Week 12, Week 16,X-ray, HE staining and scanning electron microscopy analysis were detected.Experiment three:The immunohistochemistry and Westernblot expression of BMP-2, TGF-β and IGF-1 in the bone tissue of SD rats with DM were observed in Week 12 and Week 16, and the data was statistically analyzed using different software.Results Experiment one: 1. Comparison of VAS scores of patients in both groups before and after treatment: the VAS scores of Group B(fallopia multiflora treatment group) patients before treatment in Week 12 were significantly increased in comparison with those in Week 0 and Week 4; and the differences were statistically significant(P<0.05), in terms of intragroup comparison, the VAS scores of Group B(fallopia multiflora treatment group) at Week 12 after treatment were significantly increased in comparison with those of Group A(foundation treatment group)(P<0.05).2. Comparison of QOL scores of patients in both groups before and after treatment: the QOL scores of Group B(fallopia multiflora treatment group) patients before treatment in Week 12 were significantly increased in comparison with those in Week 0 and Week 4.The QOL scores of Group A(foundation treatment group) in Week 12 comparison with in Week 0 the differences were statistically significant(P<0.05); in terms of intragroup comparison, the QOL scores of Group B(fallopia multiflora treatment group) at Week 12 after treatment were significantly increased in comparison with those of Group A(foundation treatment group)(P<0.05).3. Comparison of SF-MPQ scores of patients in both groups before and after treatment: the SF-MPQ scores of Group B(fallopia multiflora treatment group) patients before treatment in Week 12 were significantly increased in comparison with those in Week 0 and Week 4, and the differences were statistically significant(P<0.05). The SF-MPQ scores of Group A(foundation treatment group) in Week 12 comparison with in Week 0 the differences were statistically significant(P<0.05); in terms of intragroup comparison, the SF-MPQ scores of Group B(fallopia multiflora treatment group)compared with Group A(foundation treatment group)presented statistical significance after 12 weeks(P<0.05), indicating that the degree of pain alleviation in Group B was superior to that in Group A.4. Comparison of peripheral skin numbness before and after treatment in patients of both groups: the degree of numbness in patients of Group B(fallopia multiflora treatment group) in Week 8 and Week 12 presented significant difference in comparison with that on Week 0 and Week 4(P<0.05); the degree of numbness in patients of Group A(foundation treatment group) in Week 8 and Week 12 presented significant difference in comparison with that on Week 0 and Week 4(P<0.05); in terms of intragroup comparison, the numbness scores of Group B(fallopia multiflora treatment group)compared with Group A(foundation treatment group)presented statistical significance after 12 weeks(P<0.05).5. Comparison of curative efficacy in both groups: the comparison of curative efficacy in both groups in Week 12 of treatment indicated 5 excellent cases(20.00%), 11 effective cases(44.00%) and 9 ineffective cases(36%) cases in the 25 cases of the basic treatment group, and the total effective rate was 64.00%. As for the 25 cases of the Fallopia multiflora treatment group, there were 8 excellent cases(32.00%), 13 effective cases(52.00%) and 4 ineffective cases(16.00%), and the total effective rate was 84.00%. The total effective rate was prior to that of the control group and presented statistical significance(P<0.01).6. Determination result of glycosylated hemoglobin(GHb): the result of glycosylated hemoglobin in patients of Group B(fallopia multiflora treatment group) in Week 8 and Week 12 presented significant difference in comparison with that on Week 0 and Week 4(P<0.05); patients of Group A(foundation treatment group) in Week 12 presented significant difference in comparison with that on Week 0(P<0.05); The rest of the group has no obvious statistical significance.7.Comparison the change of serum phosphorus, Serum calcium, and calcium-phosphorus product of patients in both groups before and after treatment: Serum calcium and phosphorus of both groups were normal before and after treatment, difference was no statistically significant(P>0.05). Calcium-phosphorus product: difference was no statistically significant(P>0.05).8.serum AKP(alkaline phosphatase):serum AKP level of Group B in week 12 and week 8 presented significant difference in comparison with that on Week 0(P<0.05).serum AKP level of Group A in week 12 presented significant difference in comparison with that on week 0(P<0.05). The rest of the group has no obvious statistical significance.9.serum BGP(osteocalcin):serum BGP level of Group B in week 12 and week 8 presented significant difference in comparison with that on Week 0(P<0.05). serum BGP level of Group A in week 12 presented significant difference in comparison with that on week 0(P<0.05). The rest of the group has no obvious statistical significance.10.bone densitometry: difference was no statistically significant(P>0.05).Experiment two: 1.general condition: 1.1 middleweight measurement: Group B(diabetes mellitus group) compared with Group A(blank control group), Group C(insulin group) and Group D(fallopia multiflora group) presented statistical significance in week 12(P<0.05).1.2 blood glucose measurement: Group B(diabetes mellitus group, Group C(insulin group) and Group D(fallopia multiflora group) compared with Group A(blank control group) presented statistical significance in week 8(P<0.05). Group B(diabetes mellitus group) compared with Group A(blank control group), Group C(insulin group) and Group D(fallopia multiflora group) presented statistical significance in week 12 and week 16(P<0.05).1.3 glycosylated hemoglobin measurement: Group B(diabetes mellitus group) compared with Group A(blank control group) and Group D(fallopia multiflora group) presented statistical significance in week 16(P<0.05).In the terms of intraclass comparison, Group B(diabetes mellitus group) in week 16 presented significant difference in comparison with that on week 0(P<0.05).2. Blood lipid metabolism The total cholesterol, triglycerides, HDL-C and LDL in serum at week 8 from blank control group were measured, and the difference was statistically significant P<0.05 when comparing control group with insulin group, fallopia multiflora group and diabetes group; the difference was statistically significant P<0.05 by comparing blank control group at week 12 with fallopia multiflora group and diabetes group. The difference was statistically significant P<0.05 by comparing the blank control group at week 16 with insulin group, fallopia multiflora group and diabetes group. The difference was statistically significant P<0.05 by comparing diabetes group at week 16 with the same group at week 0.3. Related biochemical parameters Calcium content in serum: The difference was statistically significant(P<0.05) by comparing diabetes group at week 16(group B) with insulin group(group C), blank control group(group A) and fallopia multiflora group(group D); the difference was statistically significant(P<0.05) by comparing insulin group(group B) at week 16 with same group at week 0.Phosphorus content in serum: No statistically significant by comparing between any group at any phase(P>0.05). Calcium-phosphorus product: The difference was statistically significant(P<0.05) by comparing diabetes group at week 16(group B) with fallopia multiflora group(group D); while the difference was less statistically significant(P<0.05) by comparing insulin group at week 16 with same group at week 0; no statistically significant by comparing between any group at any phase(P>0.05).Alkaline phosphatase(AKP) content change in serum: The difference was statistically significant(P<0.05) by comparing blank control group at week 8(group A) with diabetes group(group B), insulin group(group C) and fallopia multiflora group(group D); while difference was statistically significant(P<0.05) by comparing insulin group(group B) at week 16 with insulin group(group C), blank control group(group A) and fallopia multiflora group(group D). Difference was also statistically significant(P<0.05) by comparing the insulin group(group B) at week 16 with the same group at week 12 as well as week 0.bone-Gla-protein(BGP) content change in serum: The difference was statistically significant(P<0.05) by comparing blank control group at week 8(group A) with diabetes group(group B), insulin group(group C) and fallopia multiflora group(group D). However, difference was statistically significant (P<0.05) by comparing the insulin group(group B) at week 16 with insulin group(group C), blank control group(group A) and fallopia multiflora group(group D). Difference was statistically significant(P<0.05) by comparing diabetes group(group B), insulin group(group C) and fallopia multiflora group(group D) at week 8 with that at week 0. Difference was statistically significant(P<0.05) by comparing diabetes group(group B) at week 16 with the same group at week 0.4. Dual energy X-ray: measurement of Lumbar spine and hip bone mineral density, difference was statistically significant(P<0.05) by comparing blank control group at week 8(group A) with diabetes group(group B), insulin group(group C) and fallopia multiflora group(group D). Difference was statistically significant(P<0.05) by comparing diabetes group(group B) at week 16 with same group at week 0; difference was statistically significant(P<0.05) by comparing insulin group(group B) at week 16 with insulin group(group C), blank control group(group A) and fallopia multiflora group(group D). Difference was statistically significant(P<0.05) by comparing diabetes group(group B), insulin group(group C) and fallopia multiflora group(group D) at week 8 with that at week 0.5. Light microscopy of bone tissue: the blank control group: at week 12 and 16: bone trabecular structures were evenly distributed, legible bone cells, normal bone lacunae, regular bone nucleus, and they were arranged in the middle of bone lacunae(central type). Diabetes group at week 12: visible karyopyknosis of bone cells, bone trabecular thinning and disorganization, visible irregular bone nucleis such as formation of triangle, rod-shaped or crescent-line, and nucleus were arranged along the edge of bone lacuna(peripheral type). At week 16: Bone cells were further reduced, partially karyopyknosis of bone cells, bone trabecular thinning and disorganization, irregular bone nuclei such as formation of triangle, rod-shaped or crescent-line, and nucleus were arranged along the edge of bone lacunae(peripheral type). Insulin group at week 12: Bone cells were reduced, partially karyopyknosis of bone cells, comparably large number of empty bone lacunae, thickening of the peripheral sheath, reduced and shortening of bone canaliculi, slightly thinning and disorganization of bone lacunae, irregular bone nuclei such as formation of triangle, rod-shaped or crescent-line, and nucleus were arranged along the edge of bone lacunae(peripheral); Insulin group performed similar to blank control group at week 16. Fallopia multiflora group at week 12: bone trabeculae have evenly thickness and regularly arranged, legible bone cells, normal number of bone lacunae, regular bone nucleus, and nuclears were arranged in the middle of bone lacunae(central type). Fallopia multiflora group at week 16 performed similar to the blank control group at week 16.6. Scanning Electron Microscope observation: blank control group: at week 12 and week 16, bone lacunae of the femoral neck have equal thickness and regularly arranged, spaces between bone lacunae were little, and the surfaces were smooth. Diabetes group: at week 12, the cortical bones of femoral neck of rats were thin, bone lacunae were thinner and irregularly arranged, loose three-dimensional network structures connected among bone lacunae were increased, spaces were observed between large bone lacunae, and the surfaces were slightly rough; At week 16, collagen fibers on surface of bone lacunae were irregularly and loosely arranged, a gradually rising number of empty bone lacunae, thickening of the peripheral sheath of bone lacunae, reduced and shortening of bone canaliculi, and the surfaces were rough. Insulin-treated group and fallopia multiflora treated group: at week 12, the cortical bones of the femoral neck of rats were thin, loose three-dimensional network structures connected among bone lacunae were increased, spaces were observed between large bone lacunae, and the surfaces were slightly rough. Insulin treated group at week 16: the cortical bones of femoral neck of rats were thicken, bone lacunae have equal thickness and well arranged, three-dimensional network structures connected among bone lacunae were increased, spaces were observed between large bone lacunae, and the surfaces were smooth. Fallopia multiflora group at week 16 performed similar to the blank control group.Experiment 3: as the result of immunohistochemistry and immunoblotting shown, diabetes group at week 16 illustrates significantly lower contents of BMP-2、TGF-β1 and IGF-1 than all other groups, and this result has statistically significance(P<0.05); the comparison between blank control group with other 3 groups has statistically significance(P<0.05); fallopia multiflora group at week 16 has higher contents of BMP-2、TGF-β1 and IGF-1 than when it was in week 12, and the difference is statistically significant(P<0.05)Result 1. fallopia multiflora can significantly improve clinical symptoms of the patient with diabetic osteoporosis.2. fallopia multiflora can stabilize glycosylated hemoglobin, reduce BGP and AKP contents in blood of patient with diabetic osteoporosis, and it has no obvious side effect.3. fallopia multiflora can improve blood Glucose and glycosylated hemoglobin in DOP experimental rat models.4. fallopia multiflora can change serum calcium back to normal level, increase bone density through stabilizing AKP and BGP in DOP experimental rat models.5. fallopia multiflora can recover disorders in serum lipid in DOP experimental rat models.6. Diabetic rats have decreasing contents of BMP-2, IGF-I and TGF-β1 in their bones over time.7. Both fallopia multiflora and insulin can improve the contents of BMP-2,IGF-I and TGF-β1 in their bones in diabetic rat and stimulate the formation of bones. But treatment of fallopia multiflora combined with insulin performs better than single insulin treatment. |
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