| Background and purpose.Tendons are important tissues that connect muscles and bones in the human body,with important functions of transmitting power and protecting joints.With the influence of various factors such as sports and trauma,tendon injury has become a common injury.Currently,the clinical treatment of tendon injury has been very mature,including improving tendon suture methods,improving surgical methods,using biofilms to isolate tendons,and postoperative rehabilitation treatment.However,problems such as decreased tendon healing strength,tendon adhesion formation,and re rupture still exist during the tendon healing process.Therefore,the mechanism of cytokine action in tendon healing is studied,It is necessary to find more effective treatment methods.Some studies have shown that during the tendon healing process,transforming growth factors-β3(TGF-β3)can promote the proliferation and differentiation of tendon cells,stimulate the synthesis of collagen,and participate in the regulation of tissue fibrosis.As an important intracellular transcription factor,c AMP responsive element binding protein-1(CREB-1),plays an important role in the process of tissue fibrosis,and studies have found that CREB-1 can activate TGF-β3 expression and participate in TGF-β3 regulation of downstream proteins.Other studies have shown that both TGF-β and CREB play a role in the regulation of inflammation during tendon healing.Therefore,CREB-1 and TGF-β3 may affect tendon healing and scar formation through signal pathways such as CREB-1/TGF-β3,TGF-β/Smads,but the specific mechanism of the CREB-1/TGF-β3 signaling pathway in tendon healing is not fully understood.Tendon stem cells(TSCs)are ideal cells for repairing tendon injuriesand play a key role in tendon healing.Therefore,this study explored the role of regulating the CREB-1/TGF-β3 signaling pathway in tendon healing and its possible impact on TSCs,with a view to applying it to the treatment of patients with tendon injury in clinical.Menthods.1.SD rats were randomly divided into four groups: 1,2,4,and 6 week groups.The left hind limb was used to establish the Achilles tendon injury model,and the right hind limb was used as a control.We observed the motor function and change rules of rats through gait;evaluated the tendon healing through gross observation.The injured tendons were obtained for HE staining,immunohistochemical staining,and PCR to detect the expression of related cytokines.2.The flexor tendon injury model was established in both hind limbs in c57/bl6 mouse.We construct the CREB-1 gene overexpression virus vector,the CREB-1 gene interference virus vector and a blank virus vector,and respectively inject them into the tendon injury site.They were divided into CREB-1 gene overexpression group(overexpression group),CREB-1gene interference group(interference group),virus blank vector group(negative group),and blank control group(blank group).At 1,2,4,8 weeks after surgery,the motor function and changes of the mouse were evaluated through gait observation;Tendon healing and adhesion were evaluated tthrough gross observation;At 2,4,8 weeks,the injured tendons were harvested for HE staining to observe the proliferation of tissue cells;The protein expression of TGF-β1,TGF-β3 and CREB-1 were observed by the immunohistochemical staining and collagen expression were observed by Sirius red staining3.The mouse primary tendon stem cells(TSCs)were cultured by enzymatic digestion and identified by flow cytometry.TSCs were transfected with CREB-1 gene virus vector and divided into CREB-1 gene overexpression group(overexpression group),CREB-1 gene interference group(interference group),virus vector negative group(negative group),and blank control group(blank group).The m RNA expression of CREB-1 and TGF-β3 in TSCs were detected by q PCR;The protein expression of TGF-β1,TGF-β3,CREB-1 and COL-I/Ⅲ were detected by immunohistochemistry and Western Blotting(WB).Results.1.Compared with normal controls,all gait indexes(step cycle,single stance time and average speed)were greatly affected following Achilles tendon injury,which however improved with time.The step cycle was signifificantly lower at 1,2 and 4 weeks after tendon injury(compared with normal controls,all p < 0.05),but almost returned to the normal level at 6 weeks((0.694 ± 0.102)vs.(0.503 ± 0.094)s,p > 0.05).The single stance time of the operation group was signifificantly shorter at 1 and 2 weeks after operation((0.078 ±0.010)s at 1 week,(0.078 ± 0.020)s at 2 weeks,all p < 0.001)and revealed no signifificant difference at 4 weeks(p = 0.120).The average speed of operation group at 1,2,4,6 weeks was signifificantly lower than the normal control group(all p < 0.001).2.Gross observation showed that the grade of local scar adhesion in operation group increased signifificantly at 2,4 and 6 weeks,extensive adhesion was formed at 6 weeks after operation.The results of HE staining showed that the number of fibroblast increased gradually and arranged more orderly in operation group at 1,2 and 4 weeks(all p < 0.001),and decreased at 6 weeks,but it was still signifificantly higher than the sham operation group(p < 0.001)3.Immunohistochemistry(IHC)results showed that the positive expression of TGF-β1、TGF-β3、CREB-1 in the operation group was higher than the sham operation group at all time points(all p<0.05),thich reached the peak at 2 weeks after operation,and decreased at 4weeks compared to 2 weeks(p=0.002 for TGF-β1 group,p<0.001 for TGF-β3 group,p=0.041 for CREB-1 group).PCR results showed that the m RNA expression of TGF-β1,TGF-β3 and CREB-1 in the operation group was significantly higher than the shamoperation group(all p<0.05),which reached the peak at 2 weeks and decreased at 4 weeks,and significantly decreased at 6 weeks compared to 4 weeks(all p<0.001).4.In the animal experiments,the CREB-1 overexpression group exhibited better gait behavior during tendon healing than the interference group.As the tendon healed,the step cycle gradually shortened,but the interference group was significantly longer than the other experimental groups at each time point(all p<0.05).Compared with the negative group and the blank group,the step cycle of the overexpression group was significantly shortened at1,2,4 weeks after operation(all p<0.05),but there was no statistically significant difference at8 weeks after operation(p>0.05).The average speed was the lowest 1 week after operation and gradually increased with healing time,but the speed of the interference group was lower than the other experimental groups at each time point(all p<0.05)5.By grading tendon adhesion,it was observed that the degree of adhesion gradually increased in each experimental group with the extension of the study time.But compared with the negative group and the blank group,the degree of adhesion in the overexpression group was lower,with significant differences at 2 and 4 weeks(all p<0.05).The degree of adhesion in the interference group was higher than the negative group and the blank group,with significant differences at 2 and 4 weeks(all p<0.05).In the interference group,extensive adhesion was formed at 4 weeks after operation,while in the overexpression group,some tendons did not form extensive adhesion at 8 weeks.6.The HE staining results showed that the number of fibroblasts in each experimental group first increased and then decreased with the tendon healing.The number of cells in the overexpression group was lower than the other experimental groups at each time point(all p<0.05).The interference group was higher than other experimental groups at each time point(all p<0.05).Immunohistochemical results showed that the protein expression of TGF-β3 and Smad7 in the overexpression group was significantly higher than the other experimental groups at all time points(all p<0.05),while the interference group was significantly lower than the other experimental groups at all time points(all p<0.05).The expression of Smad3 and COL-I/III in the overexpression group was significantly lower than the other experimental groups at each time point(all p<0.05).The results of Sirius red staining showed that the expression of COL-Ⅲ in tendon tissue gradually increased with the tendon healing.In addition,it was found that the expression of COL-Ⅲ was the lowest in the overexpression group at different time points,while the expression of COL-Ⅲ was the highest in the interference group.7.The immunohistochemical results of TSCs suggest that the expression levels of Smad3 and COL-I/III in the overexpression group were lower than the interference group,negative group and blank group(all p<0.05),while the expression level in the interference group was higher than the other groups(all p<0.05).The expression of CREB,TGF-β3 and Smad7 in the overexpression group were significantly higher than the other experimental groups(all p<0.05).WB results showed that the protein expression level of TGF-β3 and CREB-1 in the overexpression group was significantly higher the interference group(all p<0.05).And the interference group was significantly lower than the other groups(all p<0.05).The expression of TGF-β1 and COL-I/Ⅲ in the overexpression group was significantly lower than the other experimental groups(all p<0.05).The expression of TGF-β1 and COL-I/Ⅲ in the interference group was significantly higher than the other groups(p<0.05).Conclusions.1.In the Achilles tendon healing process,gait behavior of rat indicators are related to Achilles tendon healing.With tendon healing,gait of animal gradually improves.During tendon healing,HE staining showed that the infiltration of inflammatory cell increased in early and reached peaked at 4 weeks.This study revealed the cytokine of TGF-β1,TGF-β3and CREB-1 has certain changes law in the Achilles tendon healing process,and these cytokines may be involved in regulating Achilles tendon healing2.In animal experiments,overexpression of CREB-1 protein can reduce tendon scar adhesion,improve tendon healing and gait of animals.The infiltration of inflammatory cell increases first and then decreases during tendon healing.Overexpression of CREB-1 can reduce infiltration of inflammatory cell in tissues.Overexpression of CREB-1 can promote the protein expression of TGF-β3 and Smad7 and decrease the protein expression of Smad3 and COL-Ⅰ/Ⅲ,and reduces the ratio of COL-Ⅲ/Ⅰ.3.In the TSCs,overexpression of CREB-1 can promote m RNA expression of TGF-β3,promote the protein expression of TGF-β3 and Smad7,and reduce the protein expression of TGF-β1,Smad3 and COL-Ⅰ/Ⅲ.Therefore,there is a certain connection of tendon healing and CREB-1/TGF-β3,and the mechanism of CREB-1/TGF-β3 signaling pathway affecting tendon healing needs further research. |
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