| Objective: From the perspective of TGF-?1,to explore the effect and mechanism of halofuginone,administered by oral gavage,on osteoarthritis.1)To compare the expression of TGF-?1 in articular cartilage and subchondral bone between two groups,where articular cartilage is either intact or partial degenerated.To explore the possible role of TGF-?1 in osteoarthritis.2)Halofuginone was given orally in ACLT OA mice.From the perspective of articular cartilage and subchondral bone,to explore the role of TGF-?1 in osteoarthritis and the effect and mechanism of halofuginone on osteoarthritis.Methods: 1)To select two kinds of specimen,where articular cartilage is either intact or partial degenerated,by observing the morphology of human tibial plate.Safranin O-Fast Green staining,OARSI-modified manking score and the expression of MMP-13 were applied to assess the severity of articular cartilage degeneration.Micro-CT was used to analyze the microarchitecture of subchondral bone.Immunohistochemistry was carried out to analyze the expression of TGF-?1 in articular cartilage and subchondral bone.2)To select the appropriate dosage of halofuginone,we performed a single-dose acute toxicity test in C57BL/6 male mice of 3-month-old and calculated the median lethal dose(LD50)using the method of Bliss.C57BL/6 male mice of 3-month-old were divided into the Sham group,vehicle-,and HF-treated ACLT groups randomly.Halofuginone at three different doses or an equivalent volume of distilled water was administered by oral gavage every other day for 30 days after postoperative day 2.The mice were euthanized 30 and 60 days postoperatively.Safranin O-Fast Green staining and OARSI-modified manking score were applied to assess the severity of articular cartilage degeneration.HE staining was applied to analyze the thickness of hyaline cartilage and calcified cartilage.Immunofluorescence was applied to analyze the expression of MMP-13,Col X,TGF-?1 and p-Smad2/3 in articular cartilage.1% ITS was added to induce the chondrogenic differentiation of ATDC5 cells.Alcian Blue staining and real-time PCR were applied for chondrocyte identification.CCK-8 was used to assess the toxicity of halofuginone on chondrocyte.Western Blot was performed to explore the effect of halofuginone on TGF-?1 signaling in chondrocytes.3)C57BL/6 male mice of 3-month-old were divided into the Sham group,vehicle-,and HF-treated ACLT groups randomly.Halofuginone of 0.25mg/kg or an equivalent volume of distilled water was administered by oral gavage every other day for 30 days after postoperative day 2.The mice were euthanized 30 and 60 days postoperatively.Micro-CT was used to analyze the subchondral bone microarchitecture.Angiography that based on micro-CT was performed to assess the vessel condition in subchondral bone.TRAP staining was performed to analyze the bone resorption condition in subchondral bone.Immunohistochemistry was carried out to analyze the expression of osterix,p-Smad2/3 and CD31 in subchondral bone.Results: 1)Safranin O-Fast Green staining and OARSI-modified manking score showed that the severity of articular cartilage degeneration was more in articular cartilage(AC)partial degeneration group than that of intact(p<0.01).The expression of MMP-13,TGF-?1 and p-Smad2/3 in AC was more in AC degeneration group than that of intact(p<0.01).The expression of p-Smad2/3 in subchondral bone was more in AC degeneration group than that of intact(p<0.01).Bone volume fraction and thickness of subchondral bone plate were higher,where trabecular pattern factor was lower in AC degeneration group than that of intact(p<0.01).2)The LD50 of halofuginone in C56BL/6 male mice was calculated as 3.7514 mg/kg,and the test doses used were 0.1,0.25,and 0.5 mg/kg to investigate whether HF attenuated OA progression following oral gavage.Compared with sham group,Safranin O-Fast Green staining and OARSI-modified manking score showed that the severity of articular cartilage degeneration was more in vehicle-treated ACLT group(p<0.01),where proteoglycan loss in the halofuginone-treated group was comparable to that of the Sham group(p>0.05).The difference in AC degeneration among halofuginone groups of different dosages was not statistically significant(p>0.05).HE staining showed that the difference in the thickness of hyaline cartilage and calcified cartilage among different groups were not statistically significant 1m postoperatively(p>0.05).The thickness of hyaline cartilage was smaller,whereas thickness of calcified cartilage was bigger in vehicle-treated ACLT group than other groups 2m postoperatively(p<0.01).The difference in the thickness of hyaline cartilage and calcified cartilage among halofuginone groups of different dosages were not statistically significant 1m and 2m postoperatively(p>0.05).Compared with sham group,the expression of MMP-13,Col X,TGF-?1 and p-Smad2/3 in articular cartilage were higher in vehicle-treated ACLT group 1m postoperatively,whereas halofuginone normalized the expression aforementioned(p<0.05).Alcian blue staining showed that dye staining became deeper as time of chondrogenic induction increased.Real-time PCR showed that the expression of Col 2a1 began to increase since Day 1 and peaked at Day 14,whereas Col 10a1 transcription gradually increased and surpassed the expression of Col 2a1 by Day 21.CCK-8 revealed that the toxicity of halofuginone on chondrocytes was dosage-dependent and time-dependent.The cell viability was less than 75% when the concentration of halofuginone was more that 50ng/ml.Western Blot showed that halofuginone inhibited TGF-?1 signaling in chondrocytes in dose-dependent and time-dependent way.3)Compared with sham group,TV and trabecular pattern factor were higher,whereas the bone volume fraction and thickness of subchondral bone plate were less in vehicle-treated ACLT group and halofuginone normalized the microarchitecture of subchondral bone 1m and 2m postoperatively(p<0.05).Compared with sham group,the expression of TRAP positive cells,osterix positive cells and p-Smad2/3 positive cells were higher in vehicle-treated ACLT group(p<0.01).Halofuginone not only lowered the expression of TRAP positive cells,osterix positive cells and p-Smad2/3 positive cells,but also normalized the location of most osterix-positive cells to the bone surface,instead of the bone marrow,as was observed in the vehicle-treated group.The angiography results showed that the number and volume of vessels in subchondral bone was higher in vehicle-treated ACLT group when compared with other groups(p<0.05).Compared with sham group,the expression of CD31 positive cells were higher in vehicle-treated ACLT group(p<0.01),whereas the difference was not statistically significant between sham group and halofuginone-treated ACLT group(p>0.05).Conclusion: 1)The microarchitecture of subchondral bone changed and the expression of TGF-?1 was higher in both articular cartilage and subchondral bone when the articular cartilage was degenerated in human tibial plate.2)ACLT mice model was a reliable OA mice model.The knee joint was destabilized in ALCT mice model and the expression of TGF-?1 was elevated in articular cartilage,leading to the over-expression of MMP-13 and degradation of Col II,resulting in the degeneration of articular cartilage.Halofuginone,administered by oral gavage,inhibited the elevated activity of TGF-?1 in articular cartilage to alleviate osteoarthritis and protect the articular cartilage.3)In ACLT mice model,halofuginone,administered by oral gavage,inhibited the elevated activity of bone resorption,TGF-?1 and bone formation in subchondral bone,coupled bone remodeling and inhibited abnormal vessel formation in subchondral bone,maintained the microarchitecture of subchondral bone and protected articular cartilage. |
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