The Study On Protective Effect Of Muscone On Spinal Astrocyte And Acue Spinal Cord Injury In Rats

PART1THE PROTECTIVE OF MUSCONE ON THE PRIMARYSPINAL ASTROCYTE INJURYObjectiveTo establish the astrocyte machinery-chemical damage model, and toevaluate effects of different concentrations of muscone to astrocytedamage.MethodsAstrocytes were separated and purified by the method of short-termexposure combining with differential sticking wall. Astrocyte wasidentified by glial fibers acidic protein(GFAP) selecting cellimmunochemical staining.Astrocyte mechanical damage model(AMDM) isestablished by scratching cultured astrocytes with line2mm interval, andthe astrocyte mechanical-chemical damage model (AMCDM) is establishedby the astrocyte mechanical model adding500mmol/l glutamate.24h afterestablishing the model, LDH leakage rate and cell mortality were detectedwith trypan Blue staining method. Astrocytes were divided into group A (pure culture), group B (AMCDM), group C (AMCDM adding3ug/mlmuscone), group D (AMCDM adding6ug/ml muscone), group E(AMCDM adding12ug/ml muscone). MTT test was detected at6h,12h,24h,48h and72h.SOD, MDA, LDH, TNF-alpha and intracellular Ca2+wasdetected at3h,6h and12h.ResultsHigh purity primary spinal astrocytes were obtained by using themethod of differential sticking wall combining with short-term exposure.The mechanical-chemical damage model of the primary spinal astrocytewas established. Death cell was not discovered in group A after cultured24h, and mortality rates was23.57%in mechanical damage model, and36.27%in chemical mechanical damage model. Reducing the damagedegree of cells in groups was detected by MTT, and related with drugconcentrations. The time of maximum effect of different concentration isdifferent. At24h, LDH leakage increased significantly in group B thangroup A, and leakage increased with the time going on. Changes was notobvious in group A, LDH leakage was suppressed group C, D and E,inhibition rate of LDH in group D was88.27%. SOD detection displayedno obvious change in group A, declining obviously in group B, promotingin treating groups than group B, most obviousness in group D, at the sametime. MDA detection displayed no obvious change in group A, increasinggroup B than group A, and obvious declining group C, D and E than group B, most obvious in group D, at the same time. Changes of TNF-alpha andintracellular calcium were similar to MDA, group C, D and E and group Aand B had significant difference (P <0.05).ConclusionThe mechanical-chemical damage model of the primary spinalastrocyte was established successfully. Protective effect of differentconcentrations of muscone displayed on astrocyte damage, and protectiveeffect of6ug/ml muscone was best.PART2TO DISCUSS THE PROTECTIVE MECHANISM OF ONPRIMARY SPINAL ASTOCYTESObjectiveTo study the protective mechanism of muscone on primary spinalastrocytes.MethodsGroups divided into group A (group C adding6ug/ml muscone);Group B(AMCDM); Group C(AMDM); D group(group B adding6ug/mlmuscone); E group (group B adding50umol/l MK801); F group (group Dadding MK801). Glutamate concentration in supernatant was detectedrespectively at3h,6h and12h by ELISA method, and intracellular calciumconcentration was detected respectively at3h,6h and12h by flowcytometry method. MRNA and protein expression of EAAT, ERK1/2andGFAP were detected by Q-PCR and protein expression of those by western blot at6h. And pathological changes were observed at6h,12h,24h and48h.ResultsAt the three time points, glutamate concentration in group A was lowerthan group C, the highest group B, Lower group D than group B, lowergroup E than group B, but higher than group D, lower the F group thangroup B, D and E (P<0.001). Intracellular calcium by flow cytometrymethod in group A was lower than group C, the highest group B, lowergroup D than group B, lower group E than group B, but higher than groupD, lower group F than group B, D and E at3h,6h,and12h.(at3h,6h,P<0.05,12h, P<0.0001). MRNA expression of EAAT, ERK1/2and GFAPdetected by QRT-PCR. The ratio of EAAT mRNA in group A was lowerthan group C, the highest group B, Lower group D than group B, lowergroup E than group B, but higher than group D, lower group F than groupB, D and E.The ratio of GFAP mRNA group C was lower than group A, thehighest group B, Lower significantly group D than group B, lower group Ethan group B, but higher than group D, lower the F group than group B, Dand E. The ratio of ERK1/2mRNA is similar to GFAP. Protein expressionof EAAT, ERK1/2, GFAP by Western blot detection had the correspondingtrend of mRNA. GFAP staining: At6h, the scratching was obvious, andastrocyte growing of the edge was less, and cell swelling, group A and C isnot obvious, pyknosis was not observed. Cell swelling obviously and nuclear pyknosis was observed occasionally in group B, D, E and F, andnecrosis was observed occasionally in group B and E. At12h, the scratchbelt was obvious, and astrocyte growing of the edge was more, cell nucleuspycnosis and necrosis appeared group B, D, E and F, most obvious group B.At24h, the scratch belt was faintly visible, cell hyperplasia andhypertrophy of AST. were visible, a lot of the nucleus pycnosis andnecrosis Group B, nucleus pycnosis Group C, visible necrosis by scratchingand less of cell hyperplasia in Group D. Cell volume was larger andnucleus pycnosis and necrosis coexisted Group E. Cell volume increased,and nucleus pycnosis was found in Group F. At48h, hyperplasia andhypertrophy of AST and nucleus pycnosisin scratching area was visible ingroups, the lightest in group A, lighter in group C, most obvious andnucleus pycnosis and necrosis coexisting in group B, and the rest of thegroup lighter than group B.ConclusionProtective effect of muscone on astrocytes of machinery,mechanical-chemical damage was expressed by reducing the concentrationof glutamate, intracellular calcium, and expression of EAAT mRNA andprotein, and suppressing expression of ERK1/2mRNA and protein. Thusmuscone suppressed the inflammation, reduced the damage degree ofastrocytes, and suppressed expression of GFAP mRNA and protein,suppressed hypertrophy and hyperplasia of astrocytes. Part3PROCTECTIVE EFFECT OF MUSCONE ON ACUTESPINAL CORD INJURY IN RATObjectiveTo evaluate the protective effect of muscone on acute spinal cordinjury in rats.MethodsThe model of acute spinal cord injury (ASCIM) was established byusing Allen’s method.120rats were randomly divided into five groups,normal saline group (group NS), methylprednisolone group (group MP),group MO1(2.5mg/kg muscone group), group MO2(5mg/kg musconegroup), group MO3(10mg/kg muscone group). Group MO1, MO2andMO3were injected immediately and respectively2.5mg/kg,5mg/kg and10mg/kg muscone from the tail vein after ASCIM, continuing for sevendays, group MP injected30mg/kg methylprednisolone immediately, groupNS injected mount of saline solution. Three animals in each group wereselected randomly, and serum was extracted, and then SOD, MDA, IL-6,IL-1beta, TNF-a were detected by ELISA method at3h,8h,1d,3d,1w and2w after ASCIM. Three animals in each group were selected randomly forspinal cord tissue at8h,1d,3d,1w,2w and4w After ASCIM. H-E staining,Niss staining and of immunocytochemistry staining were selected, and thenumber per view observed of astrocytes were calculated. Function of hindlegs nerve was scored by the BBB method at1d,3d,1w,2w,3w and4w after ASCIM. Protein expression of bcl-2, caspase-3and GFAP detected bywestern-blot method at1d,3d,1w,2w and4w. The data were analyzed onstatistics.Results1. Acute spinal cord injury model was established successfully.2.SOD, MDA, TNF–alpha, IL-beta and IL-6were detected by ELISA method.(1) SOD was declining trend in NS group from3h to1w, rising at2w afterASCIM. SOD in MP and three groups treated with muscone escalated tothe peak at3days from3h, was significantly higher than NS group, mostobvious group MO3, declined2w. And the declining was most obvious ingroup MO1, MP followed, but group MO2was the highest, at the samepoint in time.(At3h,8h, P<0.001,1d, P<0.003;3d,1w,2w P<0.001).(2)MDA in NS group increased from3h to1w, falled2w, and was highergroup MO3than NS group at3h and8h, declined after1d, escalated to thepeak in group MP at3d, and then falled. The trend of group MO and MO2was similar to group MP, lowest in group MO2.(each point in time,P<0.001).(3) TNF-alpha rise slowly from3h to1w, declined at2w ingroup NS. The rest of four groups was lower than NS group, lowest groupMO2, appeared the peak in MP group at1d, and then slowly declined. Thepeak appeared in group MO3at8h, and then falled, at the same time point.(4) IL-beta and IL-6has a similar trend to TNF-alpha(Each point in time,P<0.001).3. Pathology observation (1) HE staining method, the extensive hemorrhage, cell edema, pyknosis below the dorsal spinal cord injury, wasobserved8h after ASCIM, which was the most obvious in NS group,followed by group MP. Hemorrhage was less in group MO2of three groupstreated with muscone, still more in the rest two groups. The cavity occurredbetween the strike zone and myelocele. With time going on, bleeding wasless, pyknosis, necrosis,dissolved appeared.And the astrocyte proliferationincreased gradually, significantly at1w, got to the top2w, and then sloweddown until4w.4. The cavity enlarged，and myelocele disappear4w ingroup NS, the scattered small cavities was still observed in group MP, thepart cavities was filled with hyperplasia of astrocytes, the cavities in threegroups treated with muscone disappeared basically, and inflammatoryreaction was the lightest in group MO2. At8h after ASCIM, nissl stainingdisplayed increased astrocytes, enlarged nissl bodies obviously, fragment,maldistribution, shrinked neurons in group NS. Nissl body was swollen andaccumulated, and still neurons shrinked relative integrity in group MP.Neurons were more and normal, and the distribution of nissl body wasnormal, proliferation of astrocytes was less in group MO2of three groupstreated with muscone. As time going on, there were a large number ofastrocytes, and neurons pyknosis, karyorrhexis, structure disappeared,replaced by the proliferation of astrocytes in group NS, there were a smallamount of remained and contracted neurons, and a large number ofproliferation of astrocytes obviously at4w. At the same time point of three groups treated with muscone, the number of neurons was more, structurewas integrity, but number of neurons still decreased, and satellitephenomenon was not obvious in group MO2. Neurons injury and satellitephenomenon in group MO1, MO3and MP was obvious between group NSand group MO2. Spinal cord dorsal horn, injuryed area appeared similarchanges.GFAP by immunocytochemistry staining method displayed hyperplasiaof astrocytes in group NS was obvious, went up to the top at155.8/vision at2w, and then slowly declined. Hyperplasia of astrocyte in group MP waslower than group NS, peaked at125.8/vision at1w, and then slowlydeclined. The increase of astrocytes was a declining trend after ASCIMuntil2w, then appeared to rise light at2w in three groups treated withmuscone.At same time point, the number of astrocytes in three groupstreated with muscone was lower than group NS and MP. Western-Blotdetection displayed caspase3, GFAP in group NS was highest, Bcl-2lowest,and caspase3, GFAP in group MO3was lowest, Bcl-2highest. The rest ofthe group in between group NS and group MO3.(There was a significantdifference between the group MP and NS and groups MO, at all time point,P <0.05).The scores were evaluated in5experimental groups by BBBscore.Changes of the function of lower limbs was not obvious, between0 grade and1grade1d and3d after ASCIM. With the time going on, thefunction of lower limbs was gradually improved,8.2group NS and13.6group MP at4w, and three groups treated with muscone was superior togroup MP and NS. Recover was the most obvious MO2group,10.0at2w,18.6(at4w.1d,3d, P>0.05, there was no statistical significance.1w,P<0.05,NS and MO1group, MO2, MO3group had significant difference,2w,P<0.05, group MP compared with group MO1has no statisticalsignificance, the comparison of the rest groups had significant difference. at3w,4w, P<0.05, NS group, the comparison between group MP and NSand the rest groups had significant difference).ConclusionMuscone of the different concentration had protective effect on acutespinal cord injury in rats by inhibiting immune inflammation, reducing theexpression of GFAP and caspase3, promoting the expression of BCL-2,alleviating nerve cell necrosis and apoptosis, and inhibiting the excessiveproliferation of astrocytes. And then it improved function of lower limbsafter acute spinal cord injury. Effect of muscone5mg/kg dose wasremarkable, and was better than MP.