| Low back pain is a public health problem with high incidence and recurrence rate on global scale,which is seriously harmful to people’s work efficiency and quality of life.Medical cost resulted from low back pain have brought enormous economic burdens to society and individuals.Seeking a safe,effective and inexpensive treatment is the problem that is addressed by the medical-related department currently.Acupuncture,as an effective,safe and non-invasive physical therapy,is widely recommended as a treatment for low back pain Acupuncture is a kind of mechanical stimulation,studying the mechanism that how this physico-mechanical stimulation transferred to biochemical signal is very important to reveal the effectiveness of acupuncture on low back pain.Lumbar multifidus muscle that located deeply of the erector spine muscle plays an important role in maintaining lumbar stability.Changes of the shape and disorders of function of lumber multifidus muscle can induce or aggravate low back pain.Therefore,maintaining the normal shape and function of the multifidus muscle is the key to preventing low back pain.Muscle satellite cells(MSCs)are stem cells to repair the injured skeletal muscle.The proliferation of MSCs is the key point for the regeneration and repair of injured skeletal muscle.Promoting the proliferation of lumbar multifidus MSCs is the core of the treatment of low back pain caused by multifidus muscle injury.At the early stage,a lot of animal experiments have been done on the mechanism of electro-acupuncture(EA)at Weizhong(BL40)point for treating low back pain caused by Bupivacaine.It is found that the EA at BL40 may promote the early regeneration and repair of injured multifidus muscle through the changes of serum interleukin 10(IL-10)and serum creatine kinase(CK),especially on the 4th day.Therefore,this experiment used animal experiments and cell experiments to observe the effect of EA at BL40 after multifidus muscle injury,and the expression of proliferation-related molecules and extracellular component of Collagen I and MMP2 of MSCs by Integrinβ1/ERK pathway during regeneration and repair process,revealing the mechanism of EA at BL40 on the regeneration and repair after the injury of the lumbar multifidus MSCs.Part I Effect of Electro-acupuncture at Weizhong on the Regeneration and Repair after Multifidus Muscle Injury and Extracellular Matrix RemodelingObjectiove:To observe the intervention effect of EA at BL40 on rat with multifidus muscle injury and expression of extracellular matrix(ECM)components Collagen I,MMP2,MyoD and Pax7,so as to discuss the possible mechanism of muscle tissue regeneration.Methods:24 male Sprague Dawley rats were randomly divided into normal group(NG),model group(MG),EA-BL40 group(EG),every group has 8 rats.Rats in MG and EG were treated with intramuscular injection with 0.5%Bupivacaine at bi-multifidus muscle of lumbar 4 and 5(4 points,100μL for every point).Rats in EG was treated with EA at BL40(2/100Hz in frequency,1-2mA in intensity),20min per treatment,once a day for three day.No EA intervention was given to the rats in NG and MG.Blood samples of the abdominal artery of rats in each group were separately collected for extracting serum,the serum were inactivated and filtrated for the following experiment.Injured multifidus muscle samples of each group was collected on the 4th day for morphological detection by HE,Masson staining to observe the collagenous fiber and the expression of MyoD、Pax7、Collagen Ⅰ and MMP2 were measured by WB.Result:(1)0.5%Bupivacaine injection induced significant morphology of the multifidus muscle.(2)Large area of blue-stained collagenous fiber was seen after Bupivacaine injection and it was reduced with EA intervention.(3)The expression of Collagen Ⅰ and MMP2:Compared with NG,the expression of Collagen Ⅰ and MMP2 in MG were increased(P<0.01,P<0.05),while in comparison with the MG,the expression levels of Collagen Ⅰ was significantly decreased(P<0.01)and MMP2 was significantly increased(P<0.01)in EG.(4)The expression of MyoD and Pax7:Compared with NG,the expression of MyoD was significantly increased(P<0.01)and Pax7 was significantly decreased(P<0.01)in MG;the expression of MyoD and Pax7 in EG were increased(P<0.01,P<0.05)than that in MG.Conclusion:EA at BL40 can promote the regeneration of multifidus muscle injury at early phase,which may be related to benignly regulate the expression of ECM components Collagen Ⅰ and MMP2.Part Ⅱ Isolation,Culture and Identification of Satellite Cells in Rat Multifidus MuscleObjective:To establish an efficient protocol for isolation,culture and identification of satellite cells in rat multifidus muscle.Methods:After deep anesthesia,lumber 4 and 5 multifidus muscles were isolated from 4 weeks postnatal female SD rat and digested with collagenase I.Then,the satellite cells were purified via differential adhesion velocity.They are characterized by the marker proteins of Pax7,Myod and MyHC through immunofluorescence staining method.Results:After isolation and purification,MSCs were found to be circular in morphology and high optical refractive.The shapde of them could become spindle after cultured 5d.The isolated MSCs had similar growth style and morphological characteristics.They expressed Pax7 at mitotic resting periods,MyoD in proliferated period and MyHC at early differentiation stage.Conclusion:Applying the enzyme digestion can successfully established a simple and efficient method of isolation,culture and identification of multifidus muscle satellite cells of rat.Part Ⅲ The relationship between ERK and Proliferation of Multifidus Muscle Satellite Cells and Extracellular Matrix Remodeling with Electro-acupuncture SerumObjective:To observe the intervention effect of EA at BL40 on proliferation of multifidus MSCs and expression of ECM components Collagen Ⅰ,MMP2,so as to discuss the relationship between ERK and Proliferation of multifidus MSCs and ECM Remodeling with EA Serum.Methods:The serum collected from part Ⅰ were inactivated and filtrated,and then were respectively applied to the Dulbecco’s Modified Eagle Media(DMEM)culturing multifidus MSCs as the same way as part Ⅱ with normal serum(NG),model serum(MG),EA serum(EG),and EA serum plus PD98059 group(EPG).Cell Counting Kit-8(CCK-8)was used to test the proliferation state of MSCs;WB was used to analyze the expression of Collagen Ⅰ、MMP2、MyoD、Pax7、ERK;RT-PCR was used to detect the mRNA expression of CollagenⅠ and MMP2.Results:(1)WB analysis showed that compared with NG,the expression of ERK of MG were decreased(P<0.01);while in comparison with MG,the expression of ERK of EG were increased(P<0.01).(2)The proliferation level of MSCs:① The result of CCK-8:Compared with NG,the proliferation level of MSCs were decreased(P<0.01);while in comparison with MG,the proliferation level of MSCs of EG were increased(P<0.01)and the level of MSCs of EPG were decreased(P<0.01);Compared with EG,the proliferation level of MSCs of EPG were decreased(P<0.01).②WB analysis of MyoD and Pax7:Compared with NG,the expression of MyoD and Pax7 of MG were decreased(P<0.01);while in comparison with MG,the expression of MyoD and Pax7 of EG were increased(P<0.01)and there is no difference in EPG(P>0.01);Compared with EG,the expression of MyoD and Pax7 of EPG were decreased(P<0.01).(3)ECM Remodeling state detected by WB and RT-PCR Compared with NG,the Collagen Ⅰ protein and mRNA expression of MG were increased(P<0.01),the expression of MMP2 mRNA of MG were decreased(P<0.01);while in comparison with MG,the Collagen Ⅰ protein and mRNA expression were decreased(P<0.01),the expression of MMP2 mRNA of EG were increased(P<0.01);Compared with EG,Collagen Ⅰ protein and mRNA expression of EPG were increased(P<0.01),the expression of MMP2 mRNA of EPG were decreased(P<0.01).Conclusion:EA serum may adjust the remodeling of ECM—improving the MMP2 level to degrade Collagen Ⅰ to accelerate the proliferation of MSCs and repair the injured multifidus muscle,ERK maybe involved in this process.Part Ⅳ The relationship between Integrinβ1 and Proliferation of Multifidus MuscleSatellite Cells and Extracellular Matrix Remodeling with Electro-acupuncture Serum Objective:To observe the intervention effect of EA serum on proliferation of MSCs and expression of ECM components Collagen Ⅰ and MMP2,so as to discuss the effect of Integrinβ1/ERK pathway on Proliferation of MSCs and ECM Remodeling with the intervention of EA Serum.Methods:The method is same as part Ⅲ,and adding a group of EA serum plus GRGDS(EGG).CCK-8 was used to test the proliferation state of MSCs;WB was used to analyze the expression of MyoD、Pax7、Collagen Β、MMP2、ERK、Integrinpβ1;RT-PCR was used to detect the mRNA expression of Collagen Ⅰ and MMP2.Result:(1)WB analysis of Integrinβ1:Compared with NG,the expression of Integrinβ1 of MG were decreased(P<0.01);while in comparison with MG,the expression of Integrinβ1 of EG were increased(P<0.01).(2)The proliferation level of MSCs:①The result of CCK-8:Compared with NG,the proliferation level of MSCs were decreased(P<0.01);while in comparison with MG,the proliferation level of MSCs of EG were increased(P<0.01)and the level of MSCs of EGG were decreased(P<0.01);Compared with EG,the proliferation level of MSCs of EGG were decreased(P<0.01).② The WB analysis of MyoD and Pax7 Compared with NG,the expression of MyoD and Pax7 of MG were decreased(P<0.01);while in comparison with MG,the expression of MyoD and Pax7 of EG were increased(P<0.01)and there is no difference in EGG(P>0.05);Compared with EG,the expression of MyoD and Pax7 of EGG were decreased(P<0.01).(3)ECM Remodeling state detected by WB and RT-PCR:Compared with NG,the Collagen Ⅰ protein and mRNA expression of MG were increased(P<0.01),the expression of MMP2 mRNA of MG were increased(P<0.01);while in comparison with MG,the Collagen Ⅰprotein and mRNA expression of EG were decreased(P<0.01),the MMP2 protein and mRNA expression of EG were increased(P<0.01);the Collagen Ⅰ protein and mRNA expression of EGG were decreased(P<0.01),the MMP2 protein expression was no difference(P>0.05)and mRNA expression was increased(P>0.05);Compared with EG,Collagen Ⅰ and MMP2 mRNA expression of EGG were no difference(P>0.05),the Collagen Ⅰ protein expression were increased(P<0.01)and MMP2 protein expression were decreased(P<0.01).(4)WB analysis of ERK:Compared with NG,the expression of ERK of MG were decreased(P<0.01);while in comparison with MG,the expression of ERK of EG were increased(P<0.01)and the expression of ERK of EGG were decreased(P<0.01);Compared with EG,the expression of ERK of EGG were decreased(P<0.01)Conclusion:EA serum may adjust the remodeling of ECM—improving the MMP2 expression to degrade Collagen Ⅰ to accelerate the proliferation of MSCs and repair the injured multifidus muscle,Integrinβ1/ERK maybe involved in this process. |
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