| Purpose:Articular cartilage injury is the basis of osteoarthritis and articular cartilage repair is a hotspot in the field of joint surgery.In this study,after combined application of Bushen Zhuangjin Decoction and glucosamine hydrochloride,the corresponding concentration of drug-containing serum was prepared to act on isolated rat knee articular chondrocytes,and the combined application of Chinese and Western medicines on chondrocyte proliferation and withering was observed.Regulation of death and related protein expression.The rat knee articular cartilage defect model was prepared.The combination of Bushen Zhuangjin Decoction and glucosamine hydrochloride was applied to rats.The histomorphological changes,related genes and protein expression during the repair of rat knee cartilage injury were observed.The value of the combined application method in the treatment of articular cartilage injury repair provides a new theoretical basis and ideas for the repair and treatment of knee cartilage injury.Material and method:This experiment is divided into two parts? Part one: effect of Bushen Zhuangjin decoction combined with glucosamine hydrochloride on proliferation and apoptosis of rat knee chondrocytesSD rats(32 rats)were randomly divided into 4 groups: normal saline(simple serum),Bushen Zhuangjin decoction(traditional Chinese medicine serum)and glucosamine hydrochloride(western medicine serum).Bushenzhuangjin decoction combined with glucosamine hydrochloride(combined with Chinese and Western serum group)was used to gavage the rats for one week,to prepare the corresponding concentration of serum containing drugs,and to act on the cultured rat knee chondrocytes(transmitted to the 4th passage)in vitro.The proliferation activity of chondrocytes was detected by mtt assay with different concentration(0.6%)and the expression of ii type collagen in chondrocytes was detected by flow cytometry(FCM).Part two: the mechanism of Bushen Zhuangjin decoction combined with glucosamine hydrochloride on the repair of cartilage defect in the knee of ratsSD rats(52 rats)were randomly divided into 4 groups,group A was normal saline(blank control group),group B was Bushian Zhuangjin Decoction(Chinese medicine group),group C was glucosamine hydrochloride(Western medicine group),D The rats were treated with Bushen Zhuangjin Decoction combined with glucosamine hydrochloride(Chinese and Western medicine combined group).The rats were treated with MEW.The gross repair of knee cartilage defects in 4W,8W and 12 W rats were observed by gross observation,HE staining and scanning electron microscopy.RT-The expression of collagen II and Aggrecan gene were detected by PCR.The expression of collagen II was detected by Western Blot.Results: 1.Rat knee chondrocytes were cultured in vitro,and the cells were observed to be generally spherical under an inverted microscope,and they were triangular and polygonal after adherence.From the passage to the fourth generation,it can be observed that the cell growth rate is accelerated,and the extracellular matrix with obvious refraction is visible around the cell.The cells grow in a sheet form and can exhibit an obvious "paving stone" appearance.After staining according to the Alcian blue staining kit procedure,it can be seen that the chondrocytes are mostly polygonal,the cytoplasm is homogeneous,relatively loose,most of which can be seen in the nucleolus,a few can also see the dividing phase,the cytoplasm and the membrane are blue.Color,maintaining the stability of the phenotype.The results of detection of chondrocyte proliferation by MTT assay showed that the proliferation of rat knee chondrocytes was superior to the traditional Chinese medicine serum group,western medicine serum group and simple serum group(P<0.0001).The difference was statistically significant.The significance of setting 0.6%,1.2%,2.4% drug-containing serum concentration,the concentration of drug-containing serum in the Chinese and Western medicine group was 1.2%,the most significant effect on the proliferation of chondrocytes.2.Apoptosis rate was detected by Annexin V-FITC/PI double staining,The results of flow cytometry showed that the apoptosis rate of chondrocytes was significantly decreased in the combined serum group compared with other groups(p < 0.0001),and the difference was statistically significant.3.Western Blot detection of type II collagen expression in chondrocytes.The results showed that the expression of ii collagen in chondrocytes was significantly higher in serum group than in serum group of traditional Chinese medicine,western medicine group and simple serum group(p < 0.0001).The difference is statistically significant.4.By comprehensively observing the repair of rat knee articular cartilage defects 4W,8W,12 W.At 4W,the combination of Chinese and Western medicine(group D)showed that the cartilage defect was obviously shallow,shallow and concave,and the filler was still white.Color fiber tissue;Chinese medicine group(group B),western medicine group(group C)showed that the area of cartilage defect was reduced,but there was no significant difference,the filler at the defect was white fibrous tissue;the blank control group(group A)cartilage defect was clearly visible.The signs of repair are not obvious,and there is a slight amount of fibrous tissue filling.There is still a significant difference between each group and the surrounding normal articular cartilage tissue.At 8W,the cartilage defect of the Chinese and Western medicine combination group(D group)was basically filled,the filling was similar to the hyaline cartilage tissue,but the defect mark was still visible,and the femoral condyle showed no obvious osteophyte growth;the Chinese medicine group(group B)cartilage defect The depression became shallow and filled with white fibrous tissue.The filling was still different from the surrounding normal articular cartilage tissue.There was no obvious osteophyte growth around the femoral condyle.The western medicine group(group C)was basically filled with cartilage defects,and the filling was transparent fibrous tissue.The texture is soft,the femoral condyle is slightly wide,and there is a little osteophyte growth;in the blank control group(group A),the femoral condyle cartilage defect area is reduced,shallow,and the filler is a small amount of dark fibrous tissue,and the femoral condyle is increased inside.Wide,with a few bones growing.At 12 W,the Chinese and Western medicine combined group(group D)filled the cartilage defect,and the articular cartilage in the injured area was smooth and smooth,and integrated with the surrounding cartilage tissue;close to the normal articular cartilage tissue;the Chinese medicine group(group B)cartilage defect Basic filling,bone growth around the femoral condyle;Western medicine group(C group)cartilage defect still visible a little depression,the filling is similar to cartilage tissue,the defect surface is not smooth;blank control group(group A)femoral condyle is obviously regressed Sexual change,the defect was not significantly changed compared with 8W,and the surface of articular cartilage was uneven.The general observation results preliminarily proved that the effect of Bushen Zhuangjin Decoction combined with glucosamine hydrochloride on the repair of articular cartilage defects was significantly better than that of Bushen Zhuangjin Decoction,glucosamine hydrochloride and normal saline.5.Histopathological repair of knee articular cartilage defects at 4W,8W and 12 W was observed by HE staining.At 4W,the cartilage defect of the Chinese and Western medicine combined group(group D)was completely filled with fibrous tissue,and the cell morphology was abnormal.The surface of articular cartilage was slightly sag and not smooth;in the Chinese medicine group(group B),the cartilage defect was shallow,the cartilage surface was rough,the fibrous tissue was repaired,and the subchondral bone was repaired slightly.The western medicine group(group C)showed cartilage defect.The fibrous tissue was filled,the surface of the cartilage was rough,the morphology of the chondrocytes was abnormal,and the repaired tissue was mainly repaired by fibrous tissue.In the blank control group(group A),the cartilage defect showed obvious depression,a little fibrous tissue repair,the cartilage surface was rough and the cell morphology was abnormal.At 8W,the combination of Chinese and Western medicine(group D)showed obvious proliferation of chondrocytes in cartilage defects,clear hierarchical structure,close to normal chondrocytes,and obvious repair of subchondral bone,but there were still some defects in cartilage surface;Chinese medicine group(group B)There is a small amount of chondrocyte growth in the cartilage defect,the cartilage surface is rough,and the subchondral bone is repaired a little.In the western medicine group(group C),a few chondrocytes grow in the cartilage defect,the fibrous tissue is filled,the cartilage surface is rough,and the subchondral bone is slightly Repair;blank control group(group A)showed no obvious neonatal chondrocytes in the cartilage defect,the defect was filled with fibrous tissue,the cartilage surface was rough and uneven,and the subchondral bone was not obviously repaired.At 12 W,the combination of Chinese and Western medicine(group D)showed that the cartilage repair at the cartilage defect was close to the normal cartilage surface,the subchondral bone structure was repaired normally,the repair level was clearly visible,and the chondrocyte morphology was normal;the Chinese medicine group(group B)showed cartilage defect.The depression is obviously shallow,the surface of the cartilage is not smooth,the new chondrocytes proliferate obviously,and the arrangement is close to the normal cartilage tide line.The subchondral bone repair is better than the C group;the western medicine group(C group)can still see a little depression in the cartilage defect,repair level Disorder,visible chondrocyte growth;blank control group(group A)showed obvious unevenness of cartilage defect,cartilage surface damage,cartilage cell morphology abnormal,structural disorder.The histological changes of rat knee cartilage were observed by HE staining.The repair of articular cartilage defect by Bushen Zhuangjin Decoction combined with glucosamine hydrochloride was superior to the use of Bushen Zhuangjin Decoction,glucosamine hydrochloride and normal saline.6.RT-PCR detection of 4W,8W,12 W collagenII,Aggrecan gene protein expression in each group:The expression of collagen II protein in the combination group of Chinese medicine and western medicine(group D)at 4W,8W and 12 W was significantly higher than that of traditional Chinese medicine group(group B),western medicine group(group C)and blank control group(group A)(P<0.001).Statistical significance;Chinese medicine group(group B)compared with western medicine group(group C)(P>0.05),the difference was not statistically significant;the expression of Aggrecan protein in group 4,8W,12 W Chinese and Western medicine combined group(group D)Significantly higher than the traditional Chinese medicine group(group B),western medicine group(group C),blank control group(group A)(P < 0.001),the difference was statistically significant;Chinese medicine group(group B)and western medicine group(group C)compared(P>0.05),the difference was not statistically significant.The results showed that Bushen Zhuangjin Decoction and Glucosamine Hydrochloride could promote the expression of collagen II and Aggrecan gene in chondrocytes,while Bushen Zhuangjin Decoction combined with glucosamine hydrochloride promoted the collagenN and Aggrecan gene proteins in chondrocytes.7.Western Blot was used to detect the expression of 4W,8W and 12 W collagen II in each group: 4W,8W The expression of collagen II protein in Western medicine combined group(D group)was significantly higher than that in traditional Chinese medicine group(group B),western medicine group(group C)and blank control group(group A)(P<0.001).The difference was statistically significant;Group B)compared with Western medicine group(C group)(P>0.05),the difference was not statistically significant.At 12 W,the expression of collagen II protein in the combination group(D group)was significantly higher than that in the traditional Chinese medicine group(group B),western medicine group(group C)and blank control group(group A)(P<0.001).The difference was statistically significant.The Chinese medicine group(group B)was compared with the western medicine group(group C)(P < 0.001),and the difference was statistically significant.It indicated that Bushen Zhuangjin Decoction combined with glucosamine hydrochloride can significantly promote the expression of collagenN protein in chondrocytes,and its effect is obviously better than that of Bushen Zhuangjin Decoction and Glucosamine Hydrochloride.8.Scanning electron microscopy was used to observe the repair of the microstructure of cartilage defects in each group.The combination of Chinese and Western medicine(group D)was soft.Although the defect boundary is visible in the bone defect,the surface of the regenerated cartilage tissue is flat and basically merges with the surrounding normal cartilage tissue.The structural morphology has become normal cartilage tissue.In the Chinese medicine group(group B),the cartilage surface of the articular cartilage defect is concave.The boundary was still clear and there was collagen fiber tissue growth.The western medicine group(group C)showed that the surface of the cartilage defect was flat,but it was more concave than the normal cartilage tissue.The ridge was undulating and the damaged surface was clearly visible.The blank control group(group A)showed cartilage.The surface of the cartilage in the defect is bulged,and the regenerated cartilage tissue grows irregularly and is uneven.Conclusion: 1.The combination of Chinese and Western medicine has significantly promoted the proliferation of rat knee chondrocytes,decreased the apoptosis rate of chondrocytes,and significantly promoted the expression of collagen II protein in chondrocytes.2.The combination of Chinese and Western medicine can promote the repair of rat knee cartilage defects more effectively.Through the study of this experiment,the therapeutic value of the combination of Chinese and Western medicines on the repair of articular cartilage injury is explored,which lays a certain theory for clinical application basis. |
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