| Objective Osteoporosis（OP）combined with knee osteoarthritis（KOA）is clinically common in middle-aged and elderly women.There is no standard method for clinical treatment.The understanding of its pathological mechanism has not been unified and clear.We believe that OP and KOA For different bone diseases,and kidney deficiency and blood stasis as a common pathogenesis,it is a syndrome of different diseases,and can be treated with different diseases.Therefore,this study established a model of osteoporosis and fatigue injury knee osteoarthritis in ovariectomized rats to simulate the symptoms of osteoporosis and knee osteoarthritis in middle-aged and elderly women,and to observe the pathogenesis of the disease.Qiangbaobao was intragastrically intervened to study the effect and mechanism of Qianggubao on OP combined with KOA,and to explore the scientific basis of OP and KOA for "Treat different diseases with same medicine".Methods: 1.Construction of osteoporosis combined with knee osteoarthritis model in ovariectomized rats Female SD rats of 6 months old were selected,and ovarian osteoporosis was caused by surgical removal of ovaries.After 7 days of normal feeding,running treadmill equipment was used for running fatigue training,and adaptive training with a uniformity of 15 m/min was set.day.Then change the average speed of 30 m / min,interval 5min,2 times / d,uphill slope of 16 ° continuous training for 35 d,resulting in knee joint strain arthritis,build OP and KOA combined model.Pathological tissue sections were prepared to observe the morphological changes of knee joint synovium,articular cartilage and subchondral bone;analysis and cartilage Mankin score;detection of urine CTX-Ⅱ content and serum estradiol level;detection and analysis of the third lumbar vertebrae and femur Average bone mineral density of the neck bone;biomechanical data such as three-point bending,strength,stiffness,stress and strain of the femur were tested,and MRI was combined to identify the level of OP combined with KOA.2.Therapeutic effect of Qianggubao intervention on OP combined with KOA rats After the establishment of SD rat OP combined with KOA model,Qianggubao was given intragastric intervention.The dose was 3.5g/kg,divided into two doses in the morning and evening,and the serum estradiol level was tested at 1 and 3 months after administration;biomechanical changes such as bone density,strength,stiffness,stress and strain;knee Joint pathological histomorphology and tissue microstructure changes.To analyze the efficacy of Qianggubao intervention in OP combined with KOA rats,and to explore the feasibility of OP and KOA in different diseases.3.Molecular mechanism of Qianggubao intervention in OP combined with KOA model rats.After intervention with Qianggubao,joint fluid and cartilage tissue were collected regularly（1 and 3 months after intervention）,and the expression levels of knee arthritis factor,knee articular cartilage Wnt5 a,β-catenin,OPG,RANKL and other proteins were determined by ELISA.To analyze the effects of Qianggubao on Wnt5 a,β-catenin,OPG and RANKL m RNA in knee joint cartilage,and to explore the molecular mechanism of Qianggubao’s intervention in OP combined with KOA based on the same disease.Result: 1.Successfully established rat OP combined KOA model The analysis showed that the ovarian ablation group（OVX）and the combined running training group（OVX+RUN）,after running for 4w and 6w,compared with the blank control group,the joint fluid TNF-α,IL-1,urine The CTX-Ⅱ content was significantly changed.The E2 of OVX and OVX+RUN were significantly lower than those of the blank control group（P<0.05）,（P<0.01）,indicating that the removal of ovary significantly reduced the E2 content,and the running training would increase the E2 content.The content of each factor above 6w in running training was: OVX+RUN group: TNF-α（32.18±8.23）,IL-1（37.45±9.22）,CTX-Ⅱ（251.71±46.27）;OVX group: TNF-α（23.85±8.85）,IL-1（24.26±10.85）,CTX-Ⅱ（171.55±53.10）;BLANK group: TNF-α（11.55±4.29）,IL-1（10.43±2.79）,CTX-Ⅱ（41.55±12.03）were significantly higher than the blank control group.The content of each factor above 4W in running training was: OVX+RUN group: TNF-α（28.36±4.34）,IL-1（27.91±3.8）,CTX-Ⅱ（220.85±49.42）;OVX group: TNF-α（19.98）±4.84),IL-1（17.16±4.58）,CTX-Ⅱ（143.15±38.85）;BLANK group: TNF-α（11.37±3.81）,IL-1（9.93±2.42）,CTX-Ⅱ（57.72±14.50）.Both were significantly higher than the content of the blank control group.At the same time,the value of each factor of running 6w is significantly higher than 4w.It indicated that ovarian ablation combined with running training can significantly cause knee arthritis in rats,and the degree of inflammation is aggravated with the extension of running training time.The cartilage Mankin score found that after 4 weeks of running training,there was a significant difference between the experimental group and the Blank group（P<0.01）.The Mankin of each group were: OVX+RUN group（6.14±0.73）,OVX group（4.94±0.49）,and BLANK group（1.14±0.41）;there was significant difference between experimental group and Blank group（P<0.01）,OVX group.There are also significant differences between the OVX+RUN group and the OVX+RUN group.After 6 weeks of running training,there was a significant difference between the experimental group and the BLANK group（P<0.01）.The Mankins in each group were: OVX+RUN group（7.91±1.05）,OVX group（5.54±0.71）,and BLANK group.（1.00±0.34）;there was also a significant difference between the OVX+RUN group and the OVX group（P<0.01）.At the same time,compared with the running training 4W,whether it was the OVX group or the OVX+RUN group,the running training was 6 weeks.After that,their Mankin scores were significantly improved.It was again shown that ovarian ablation combined with running training can significantly cause knee arthritis in rats,and the degree of inflammation is aggravated with the prolonged running time.The bone density test found that after 4 weeks of running training,L3 lumbar vertebrae: OVX+RUN group（0.16±0.01）,OVX group（0.15±0.01）,and BLANK group（0.19±0.01）;right femur proximal end R1:OVX+RUN Group（0.25 ± 0.03）, OVX group（0.22 ± 0.02）,and BLANK group（0.29 ± 0.02）.There was a significant difference between the experimental group and the blank,and there was also a significant difference between the OVX and OVX+RUN groups.After 6 weeks of running training,L3 lumbar vertebrae: OVX+RUN group（0.13±0.01）,OVX group（0.14±0.01）,and BLANK group（0.19±0.00）;right femur proximal R1:OVX+RUN group（0.19±0.01）),OVX group（0.21 ± 0.01）,and BLANK group（0.30 ± 0.02）.There was a significant difference between the experimental group and the blank,and there was also a significant difference between the OVX and OVX+RUN groups.After 6 weeks of running training,the three-point bending test and bone density values of the OVX+RUN group rebounded compared with the 4th week.The above experimental results showed that the OP combined KOA rat model was successfully constructed by ovarian ablation combined with running training for 4-6 weeks.2 Effect of Qianggubao intervention on osteoporosis and knee osteoarthritis in ovariectomized rats After 1 month and 3 months of OP combined with KOA rats,the test showed that: 1 month after intervention,Qianggubao gavage group and Jingutongxiao pill gavage group were compared with saline group（P<0.05）.The E2 content of each group was: Qianggubao gavage group（49.59±8.15）,Jingutongxiao pill gavage group（48.08±16.01）,saline group（33.12±7.75）;intervention for 3 months,Qianggubao Compared with the normal saline group,the stomach group and the Jingutongxiao pill group were compared with the saline group（P<0.01,P<0.05）.The E2 content of each group was: Qianggubao gavage group（54.73±16.38）,Jingutongxiao pill gavage group（52.97±14.34）,and saline group（34.57±5.92）,and E2 content was significantly increased.Cartilage Mankin score: 1 month of drug intervention: Qianggubao group,Jingutongxiao pill group and normal saline group were compared（P<0.05）,the scores of each group were: Qianggubao gavage group（4.65±0.56）,Jingutongxiao pill gavage group（4.57±0.57）,saline group（5.48±1.01）;drug intervention for 3 months: Qianggubao group,Jingutongxiao pill group and normal saline group were compared（P<0.01）,the scores of each group were: Qianggubao gavage group（4.28±0.50）,Jingutongxiao pill gavage group（4.02±0.29）,and saline group（5.77±0.52）,all of which had curative effect.The determination of IL-1 in rat joint fluid showed that after 1 month of administration,Qianggubao group and Jingutongxiao pill group were significantly more significant（P<0.01）than normal saline group.The contents of each group were: Qianggubao group（19.66±6.70）,Jingutongxiao pill gavage group（16.97±6.02）,saline group（32.55±7.89）;after 3 months of administration,Qianggubao group and Jingutongxiao pill group The results were significantly higher（P<0.01）than those in the saline group.The contents of each group were: Qianggubao gavage group（17.85±4.87）,Jingutongxiao pill gavage group（15.24±7.26）,and saline group（30.03±）.3.86).The content of TNF-α in rat joint fluid was found to be higher than that of normal saline group after 1 month of administration: Qianggubao group and Jingutongxiao pill group compared with saline group（P<0.05,P<0.01）.The contents of each group were: Qianggubao gavage group（23.51±9.31）,Jingutongxiao pill gavage group（20.22±5.50）,saline group（34.03±8.99）;after 3 months of administration,Qianggubao group and the Jingutongxiao pill group were compared with the saline group（P<0.01）,which was higher than the saline group.The content was significantly higher than that at the time of administration,and the contents of each group were: Qianggubao gavage group（22.19±8.57）,Jingutongxiao pill gavage group（18.53±7.33）,and saline group（37.08±）.10.11);It shows that Qianggubao and Jingutongxiao Pill can inhibit the expression of IL-1 and TNF-α inflammatory factors,and the curative effect is remarkable.The content of CTX-Ⅱ in rat urine was found to be higher than that of normal saline group after 1 month of administration,Qianggubao group and Jingutongxiao pill group compared with saline group（P<0.05,P<0.01）.The contents of each group were: Qianggubao gavage group（395.12±147.82）,Jingutongxiao pill gavage group（303.39±93.15）,saline group（562.04±106.55）;after 3 months of administration,Qianggubao Both the group and the Jingutongxiao pill group were significantly more significant（P<0.01）than the saline group,and the content was significantly higher than that at the 1 month of administration.The contents of each group were: Qianggubao gavage group（333.56±88.73）,Jingutongxiao pill group（260.99±75.36）and saline group（529.43±110.55）;however,there was no statistically significant difference between Qianggubao group and Jingutongxiao pill group（P>0.05）.Bone mineral density measurement showed that the DXA measurement results of 1 month of drug intervention,L3 lumbar vertebrae bone density（g/cm2）showed: Qianggubao group,Jingutongxiao pill group and normal saline group（P<0.01,P<0.05）The upper end of the right femur R1 showed: Qianggubao group,Jingutongxiao pill group and normal saline group（P<0.01,P<0.05）.The DXA measurement results of drug intervention for 3 months,L3 lumbar vertebrae bone mineral density（g/cm2）showed: Qianggubao group,Jingutongxiao pill group and normal saline group（P<0.01,P<0.05）;right femur upper end R1 showed: Qianggubao group,Jingutongxiao pill group and normal saline group（P<0.01,P<0.05）.The DXA measurement results of drug intervention for 3 months,L3 lumbar vertebrae bone mineral density（g/cm2）showed: Qianggubao group,Jingutongxiao pill group and normal saline group（P<0.01,P<0.05）;right femur upper end R1 showed: Qianggubao group,Jingutongxiao pill group and normal saline group（P<0.01,P<0.05）.Biomechanical measurement found that L3 lumbar compression test（Compression test）showed that drug intervention for 1 month: Qianggubao group,Jingutongxiao pill group compared with saline group: maximum load（k N）,maximum stress（N/mm2）,the maximum strain（%）were both（P<0.01,P<0.05）.Drug intervention for 3 months: Qianggubao group,Jingutongxiao pill group and normal saline group,maximum load（k N）（P<0.01,P<0.05）;Qianggubao group,Jingutongxiao pill group and normal saline In the group comparison,the maximum stress（N/mm2）（P<0.01,P<0.05）and the maximum strain（%）（P<0.01）;the three-point bending stress detection of the right proximal femur R1 showed that the drug intervention was 1 month: Qianggubao group,Compared with the saline group,the maximal load（k N）,maximum stress（N/mm2）,maximum strain（%）,and fracture load（k N）were all（P<0.01,P<0.05）.Drug intervention for 3 months: Qianggubao group,Jingutongxiao pill group and saline group: maximum load（k N）,maximum stress（N/mm2）,maximum strain（%）,and breaking load（k N）（P<0.01,P<0.05）,was statistically significant.It was shown that after treatment with the drug,the Qianggubao group and the Jingutongxiao pill group were significantly higher than the saline group,but there was no statistically significant difference between the Qianggubao group and the Jingutongxiao pill group（P>0.05）.The above results indicate that Qianggubao’s intervention in OP combined with OKA is effective,and the treatment of Jingutongxiao pill has also achieved curative effect,indicating that OP and OKA are the same disease,and it is feasible to treat the same disease.3 Mechanism of Qianggubao Intervention on Osteoporosis Combined with Knee Osteoarthritis in Ovariectomized Rats After 1 and 3 months of drug intervention,the content of OPG in the articular cartilage of each group was determined.The expression of OPG protein was detected in the cartilage of each group.The intervention was 1 month.The expression of each group was: blank Group（0.62±0.14）,normal saline group（0.33±0.07）,Qianggubao group（0.50±0.13）,Jingutongxiao pill group（0.57±0.13）;administration intervention for 3 months,the expression levels of each group were respectively They were: blank group（0.71±0.19）,saline group（0.37±0.07）,Qianggubao group（0.58±0.21）,and Jingutongxiao pill group（0.66±0.19）;The expression of chondrocytes in the normal control group was the highest;the expression of cartilage in the rats of Qianggubao intervention group and Jingutongxiao pill group was significantly weakened,and the saline group（OP combined with KOA model group）was the lowest.The expression of RANKL was found to be 1 month after administration of the intervention,the expression levels of RANKL in the articular cartilage of rats were: blank group（0.30±0.10）,saline group（0.51±0.12）,Qianggubao group.（0.35±0.07）,Jingutongxiao Pill group（0.27±0.13）;the drug intervention for 3 months,the expression of each group were: blank group（0.30±0.15）,saline group（0.58±0.20）,Qianggubao group（0.31±0.10）and Jingutongxiao pill group（0.24±0.12）;the expression of cartilage in the normal control group was the lowest;the normal saline group（OP combined with KOA model group）had the highest expression;The protein expression in the cartilage tissue of the rats treated with the Jingutongxiao Pill group was higher than that of the blank control group,but there was no significant difference between the two drug intervention groups.The expression level of Wnt5 a was found to be 1.After administration of the intervention,the expression levels of Wnt5 a in the articular cartilage of rats were as follows: blank group（0.43±0.06）,saline group（0.76±0.13）,Qianggubao group（0.42±0.13）and Jingutongxiao pill group（0.37±0.13）;the administration dose was 3 months,and the expression levels of each group were: blank group（0.31±0.14）and saline group（0.61±）.0.20),Qianggubao group（0.41±0.16）and Jingutongxiao pill group（0.33±0.15）;the expression of cartilage in the normal control group was the lowest;the expression of the saline group（OP combined with KOA model group）was the highest;The protein expression levels in the rat cartilage tissue of the Qianggubao intervention group and the Jingutongxiao pill intervention group were higher than those of the blank control group,but there was no significant difference between the two drug intervention groups.The expression of β-catetin was determined.After 1 month of administration,the expression levels of β-catetin in the articular cartilage of rats were: blank group（0.36±0.13）,saline group（0.59±0.11）,strong.The bone medicine group（0.36±0.12）and the muscle bone pain elimination group（0.28±0.14）;the administration intervention for 3 months,the expression levels of each group were: blank group（0.36±0.10）,saline group（0.57±0.18）.Qianggubao group（0.32±0.10）,Jingutongxiao pill group（0.26±0.08）;the expression of cartilage in the normal control group was the lowest;the normal saline group（OP combined with KOA model group）had the highest expression;strong The protein expression levels in the cartilage tissues of the bone-Bao intervention group and the Jingutongxiao pill group were higher than those in the blank control group,but there was no significant difference between the two drug intervention groups.q PCR detection of OPG,RANKL,Wnt5 a,β-catenin gene transcription levels found: drug intervention in January and March,the two drug intervention group and saline group compared gene transcription level was significantly different（P<0.05）The difference in the transcriptional level of these genes in each group is completely consistent with the difference in the protein expression levels of these genes,indicating that Qianggubao is treated with the treatment of OP and KOA with the “Treat different diseases with same medicine”.The signal transduction pathway associated with OPG,RANKL,Wnt5 a,and β-catetin is achieved.Wnt/β-catenin is associated with osteoarthritis and osteoporosis and is involved in bone metabolism.Wnt5 a is an inflammatory mediator involved in a variety of inflammatory responses and is highly expressed in articular cartilage.It not only acts on the Wnt/β-catenin pathway,but also activates non-canonical pathways.Stimulation of Wnt in OA cartilage can lead to aggregation of β-catenin and affect metabolic disorders of arthritis.The increase in the expression of Wnt5 a is also positively correlated with β-catenin.OPG/RANKL/RANK is an important signaling pathway regulating cartilage damage and a pathway between osteoblasts and osteoclasts.The combination of OPG and RANKL inhibits bone resorption,and the combination of RANK and RANKL enhances bone resorption.The OPG/RANKL/RANK signal is a downstream part of the Wnt/β-catenin signaling pathway,and Wnt/β-catenin activation downstream OPG/RANKL/RANK can also interact.The strength of OPG/RANKL was increased by Qianggubao,and the knee cartilage was protected and repaired.In the OA combined with OP,the regulation of Wnt/β-catenin signaling pathway is particularly important for the prevention and treatment of the two diseases.Conclusion: 1.Rat bilateral ovariectomy combined with fatigue injury induced injury,can successfully establish OP combined KOA rat model,and the new model has high stability,in line with the clinical pathological changes of knee osteoarthritis in postmenopausal women.2.Qianggubao Decoction has the effects of benefiting kidney and strengthening bone,promoting blood circulation and relieving pain.It can up-regulate E2 in serum and reduce urinary type II collagen carboxy propeptide（CTX-Ⅱ）,prevent articular cartilage from accelerating lesions and effectively reduce IL.-1,TNF-α inflammatory cytokines and promote the repair of knee joint injuries.Guiding Qianggubao intervention OP combined with KOA is effective,indicating that OP and KOA are the same syndromes,and it is feasible to treat different diseases.3.Qianggubao has an up-regulation effect on OPG,which can reduce the expression of Wnt5 a,β-catetin and RANKL m RNA,indicating that Qianggubao intervenes to treat OP and KOA with “Treat different diseases with same medicine”,possibly through OPG.,RANKL,Wnt5 a,β-catetin associated signal transduction pathways are implemented.4.Qiangbao can regulate estrogen levels,inhibit osteoclasts and enhance osteoblast activity.Fangzhong: The tonifying kidney drugs have an estrogen-like effect,which can regulate the levels of estrogen and the expression of gene proteins.Blood-activating drugs can improve systemic and local blood circulation,delay the development of OP combined with KOA,and achieve the role of “Treat different diseases with same medicine” to prevent postmenopausal osteoporosis combined with knee osteoarthritis. |
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