Effect Of Didang Decoction On AQP4 Expression In The Treatment Of Acute Spinal Cord Injury In Rats Under The Guidance Of Tongfu-zhuyu Therapy

Objective This study is based on the clinical experience of the supervisor,combined with the research status and hotspot of acute spinal cord injury,through inhibiting the expression of AQP4 in spinal cord tissue of SCI rats to reduce spinal cord edema after ASCI as the entry point.The purpose of this study is to explain the possible mechanism of treating ASCI with Didang decoction under the guidance of "Tongfu-Zhuyu" therapy,so as to provide theoretical basis for treating ASCI with "Tongfu-Zhuyu" therapy.Methods Part Ⅰ30 SD rats aged 10 weeks were randomly divided into sham operation group(group A),self-made percussion device 2cm group(group B1),self-made percussion device 4cm group(group B2),NYU group 1.25 cm group(group C1)and NYU group 2.5cm group(group C2)according to random number table method,with 6 rats in each group.Except for the Sham group,the other groups were modeled with the modified Allen’s method.Groups B1 and B2 were able to hit the T10 spinal cord from a distance of 2cm and 4cm,respectively.Groups C1 and C2 were able to hit the T10 spinal cord from a distance of 1.25 cm and 2.5cm,respectively,using NYU.BBB score and Reuter score were performed on 1,3,5 and 7 days after modeling.After 7 days,paraffin-embedded sections and Nissl staining were performed to observe the degree of tissue damage and survival rate of neurons in each group.Part II60 SD rats aged 10 weeks were randomly divided into Sham operation group(Sham),Model group(Model),Didang decoction group(DDD),TGN-020 group(TGN-020)and Didang decoction combined with TGN-020group(DDD+TGN-020),with 12 rats in each group.In Sham group,only the lamina was opened to expose the spinal cord without injury.The spinal cord was hit from a height of 40 mm by a self-made spinal cord percussion device in the other groups.Sham group and Model group will not be any postoperative treatment intervention,DDD group underwent 3d to resist the Didang decoction,daily for 3d.According to the dosage of rats and surface area of conversion method,DDD group rats daily dosage is 5.04g/kg,using distilled water to dilute give lavage treatment after a certain proportion,daily 1time in the morning and night.Treatment method of TGN-020 group:intraperitoneal injection,the dose was 5mg/kg.Treatment of DDD+TGN-020group: TGN-020 was given intraperitoneal injection,followed by DDD gavage.At the end of treatment intervention,behavioral evaluation(BBB test and Reuter score)was used to observe the recovery of neurological function.After the rats were sampled,Gross observation and moisture content detection were performed on the spinal cord tissue.He staining was used to observe the degree of tissue damage,Nissl staining was used to observe the morphology of neurons,immunofluorescence was used to detect the number of AQP4 GFAP co-expressed positive cells in the spinal cord tissue of each group,and Western Blot was used to detect the AQP4 GFAP CSPG in the spinal cord tissue of each group.The expression level of PCNA NGF BDNF GAP-43 protein,the expression level of CSPG PCNA gene in spinal cord tissue of each group was detected by q PCR method,the morphology of myelin sheath and neuron tissue was observed by transmission electron microscopy,and the morphology of dendrites and dendritic spines were observed by Golgi staining.Part III40 SD rats aged 8 weeks were randomly divided into blank serum group and drug-containing serum group.The drug-containing serum group was given5.04g/kg DDD by gavage for 3 days,while the blank serum group was given equal dose of superpure water by gavage for 3 days.After the intervention,serum was extracted for use.8-12 embryonic rats were taken at a time,and astrocytes(AS)were extracted by mechanic-enzyme digestion method.The cells were cultured in 10% high glucose medium,and the cells were observed and passed to the third generation for subsequent experiments.Firstly,GFAP content in cells was identified by immunofluorescence,and then experimental groups were conducted: Blank group(the Control group,CG),Model group(Model group,MG),Blank serum group(Blank serum group,BSG),DDD drug-containing serum group(DDD),intervention group(TGN-020)the TGN-020 and DDD combined the TGN-020 group(DDD+TGN-020),in addition to MG,the rest of the group to scratch method building,building after the completion of the DDD drug-containing serum intervention,BSG isodose Blank serum intervention,TGN-020 was given with 100 n M TGN-020 intervention,DDD+TGN-020 was given with TGN-020 intervention before DDD intervention.Determined by MTT method to detect DDD drug-containing serum intervention AS the safety of the intervention rate,the concentration of the best intervention time and intervention,determined by MTT method to detect each group of RAS activity and the effect of immunofluorescence staining method to detect AQP4 in RAS and GFAP expression on the number of cells,Western Blot method to detect groups of AQP4 in the RAS,GFAP,and model NGF,BDNF,GAP-43 protein expression of the quantity,the microscopic observation of DDD drug-containing serum on the mobility of RAS.Statistical method SPSS21.0 software was used for statistical processing,and the measurement data was expressed as mean±standard error(` X±S).All data obtained were tested for normal distribution.One-way ANOVA was used if the data met the normal distribution,LSD method was used when the variance was homogeneous,and Games Howell method was used when the variance was uneven.If it does not fit the normal distribution,the non-parametric test is used.P<0.05 indicated that the difference was statistically significant.Result Part Ⅰ1.Comparison of spinal cord morphology in each group At the T10 segment of group A,the dura was complete,the spinal cord tissue was uniformly milky white,and the shape was full and round.The dura at the T10 segment in the B1,B2,C1 and C2 groups were all complete,with different degrees of bright red or dark red in color,edema and stasis and hyperplasia of scar tissue could be seen at the injury sites.Compared with the B1 and C1 groups,the edema in the B2 and C2 groups was more serious,and the range of stasis was wider and the color of deeper scar tissue was more.2.Comparison of behavioral scores The BBB score showed that group A had no abnormal function of lower limbs except the decrease of exercise amount caused by surgery,and the score was 21 points.Other groups showed different degrees of motor dysfunction,as follows: compared with B1,C2 and C1,BBB score was significantly lower,the difference was statistically significant(P<0.05);There was no significant difference in BBB scores between B1 and C1,B2 and C2(P>0.05).Reuter score showed that no abnormal sensory function was found in group A.The other groups showed different degree of sensory function abnormalities after surgery,specifically B2 and C2 group 1 day after the absence of stretch reflex,muscle strength and muscle tension weakened or disappeared,pain retraction reflex blunt,back sensation or disappearance;In the B1 and C1 groups,stretch reflex,muscle tension,pain retraction reflex and back sensation all existed except muscle strength disappeared on day 1 after surgery,and muscle strength began to recover on day 3 after surgery.The Reuter scores of B2 were significantly lower than those of B1 and C2 and C1(P<0.05).There was no significant difference in Reuter scores between B1 and C1,B2 and C2(P>0.05).3.Nissl staining The results of Nissl staining showed that the spinal cord tissue in group A had complete structure,clear boundary between gray and white matter,regular neuronal cell morphology,large nucleus,clear nucleoli and large number,and plaques like Nissl body could be seen in the cytoplasm.Compared with group A,the structural damage of other groups was more serious,with unclear gray matter structure,larger tissue gap,vacuolation of cells or cell shrinkage and volume reduction,decreased number of neurons,and blurred or disappeared Nissolith in the injured areas.4.Ratio of damaged area of spinal cord The results showed that no damaged area was found in group A,and the ratio was 0.The other groups were higher than group A,and the difference was statistically significant(P<0.05).Compared with B1 and C1,the damage area ratio of B2 and C2 was significantly higher,and the difference was statistically significant(P < 0.05).There was no significant difference in damage area ratio between B1 and B2 and between C1 and C2(P>0.05).5.Comparison of the degree of neuronal injury in anterior and posterior horn of spinal cord in each group By analyzing the number of positive neurons in anterior and posterior horn,the results showed that group A had more neurons and more Nissellite content,while other groups were less than group A,the difference was statistically significant(P<0.05).Compared with B1 and C1,the number of positive neurons in B2 and C2 decreased significantly,and the difference was statistically significant(P < 0.05).There was no statistically significant difference in the number of positive neurons between B1 and B2,C1 and C2(P>0.05).Part Ⅱ1.Gross observation of spinal cord tissue in each group From the general observation,the spinal cord tissue in the Sham group was bright white,intact,and no blood stasis or edema was observed.In the Model group,there was obvious blood stasis area at the injury site,the blood stasis was dark red,the blood stasis was large,and there was moderate edema of the spinal cord.In DDD group,small blood stasis area was found at the hit injury site,and the blood stasis area was pale red with slight edema.DDD group,TGN-020 group and DDD+TGN-020 group were similar in general observation,but no significant difference was observed by naked eyes.2.Water content of spinal cord tissue in each group Compared with Sham group,water content of spinal cord tissue in other groups was significantly increased(P<0.05);Compared with Model group,water content of spinal cord tissue in DDD group and TGN-020 group was significantly decreased(P < 0.05);There was no significant difference in water content of spinal cord tissue between DDD group and TGN-020 group(P>0.05).3.Behavioral observations BBB score,inclined plate test results and Reuter score of rats in all groups showed that,compared with Sham group,BBB score and inclined plate test score of rats in other groups were significantly decreased(P<0.05),while Reuter score was significantly increased(P < 0.05).Compared with Model group,BBB score in DDD group,TGN-020 group and DDD+TGN-020 group was significantly increased(P < 0.05),and Reuter score was significantly decreased(P<0.05);There was no significant difference between DDD group,TGN-020 group and DDD+TGN-020 group(P>0.05).4.HE staining In the Sham group,the tissue structure was complete,and the morphological structure of neurons and other cells was good.No scar tissue or cavity formation,no blood stasis area or exudation of inflammatory substances were observed.In the injury site of Model group,there were disorderly tissue structure,a large amount of scar tissue and the formation of spinal cavity,obvious blood stasis area,accompanied by a large number of inflammatory cell infiltration,and shrinkage and reduction of neurons and other cells.In the other three groups,the morphological structure of the tissue was complete,a small amount of syringomyelia was formed,the area of blood stasis was small,the inflammatory cells were clear and moist,and the morphological structure of neurons and other cells was complete.The cavity area of each group was quantitatively analyzed by using Image J software.The results showed that compared with Sham group,the cavity area of the Model group was significantly increased(P<0.05).Compared with Model group,the area of syringomyelia in DDD group,TGN-020 group and DDD+TGN-020 group was significantly decreased(P<0.05).There was no significant difference in the area of myeloid cavity between DDD group,TGN-020 group and DDD+TGN-020 group(P>0.05).5.Nissl staining The morphology and structure of the spinal cord in the Sham group were complete,and the gray matter was butterfly-like,with a clear boundary between gray and white matter,and more neurons were distributed.Place-like Nistellosis with clear staining could be seen in the cytoplasm of the neurons.The neuronal cell body was full,the nucleus was large,and the nucleoli were obvious.In the Model group,the spinal cord tissue was seriously damaged,with incomplete structure,unclear gray and white matter boundaries,a large number of inflammatory cell infiltration,neuronal shrinkage,liquefaction,necrosis,formation of more vacuoles,smaller or disappeared Nistellite.Compared with Model group,the spinal cord structure of DDD group,TGN-020 group and DDD+TGN-020 group was more complete,with faint-visible gray matter boundary,more neurons,less vacuol-like changes,more and fuller Nistellites.However,compared with the Sham group,there were still pathological changes such as pyknosis,reduced cytoplasm,smaller nucleus and reduced Nissolith.6.The expression levels of AQP4 and GFAP positive cells in each group were detected by immunofluorescence The expression of AQP4 positive cells was green,the expression of GFAP positive cells was red,the DAPI staining was dark blue,the expression of AQP4+/GFAP+ cells was yellow dendroid,the staining background was clean,the expression was clear,and the nuclear number was not significantly different(P>0.05),which was comparable.Few or no obvious AQP4+/GFAP+cells were seen in the Sham group,while more AQP4+/GFAP+ cells were seen in the Model group,with high cell brightness and severe cell swelling.Compared with model group,DDD group,TGN-020 group and DDD+TGN-020 group had fewer cells,lower brightness and less cell swelling.The AQP4+/GFAP+ cells in each group were counted and counted.Compared with Sham group,the proportion of AQP4 and GFAP positive cells in the other groups was significantly higher(P<0.05).Compared with Model group,the proportion of AQP4 and GFAP positive cells in DDD group,TGN-020 group and DDD+TGN-020 group was significantly decreased(P<0.05).There was no significant difference in the percentage of AQP4 and GFAP positive cells among DDD group,TGN-020 group and DDD+TGN-020 group(P>0.05).7.Western Blot was used to detect the protein expressions of AQP4,GFAP,PCNA,CSPG,NGF,BDNF and GAP-43 in each group The background of protein bands in each group was clear,and there was no heterozygous phenomenon.The trend of protein expression was obvious and comparable.Compared with Sham group,the gray values of AQP4,GFAP,PCNA and CSPG proteins in spinal cord tissue of all groups were significantly increased(P<0.05),and the gray values of NGF,BDNF and GAP-43 proteins were significantly decreased(P<0.05).Compared with Model group,the gray values of AQP4,GFAP,PCNA and CSPG proteins in spinal cord tissue of DDD group,TGN-020 group and DDD+TGN-020 group were significantly decreased(P < 0.05),while the gray values of NGF,BDNF and GAP-43 proteins were significantly increased(P < 0.05).There was no significant difference in the gray values of AQP4,GFAP,PCNA,CSPG,NGF,BDNF and GAP-43 in the spinal cord between the DDD group,TGN-020 group and DDD+TGN-020 group(P>0.05).8.Expression levels of PCNA and CSPG genes in spinal cord tissue of each group The amplification curves of each group were stable,and the dissolution curves were smooth and unimodal,indicating that the primers were well designed and the template was not contaminated,and the experimental results were comparable.The relative expression levels of target genes in each group showed that compared with Sham group,the expression levels of PCNA and CSPG genes in spinal cord tissue in each group were significantly increased(P<0.05).Compared with Model group,the expression levels of PCNA and CSPG genes in spinal cord tissue of DDD group,TGN-020 group and DDD+TGN-020 group were significantly decreased(P<0.05).The results of comparison between DDD group,TGN-020 group and DDD+TGN-020 group showed no significant difference in the expression levels of PCNA and CSPG genes in spinal cord tissue among all groups(P>0.05).9.The myelin sheath and neuronal tissue morphology were observed by transmission electron microscopy Under transmission electron microscope,spinal cord tissue in Sham group showed regular myelin sheath shape,uniform thickness,concentric circle shape,complete structure,no rupture,deletion and other changes were observed.The lamellar structure was uniform and dense,with abundant intracellular mitochondria and full cytoplasm,which could be clearly seen.In the Model group,the myelin sheath structure was irregular,with partial myelin sheath fracture and missing,varying thickness,local folds and curls,loose lamellar structure and fusion,mitochondria swelling,and even partial dissolution and disappearance.In DDD group,TGN-020 group and DDD+TGN-020 group,the myelin structure was more complete,and the morphology of some myelin was irregular but improved compared with that of the Model group.Some myelin sheath was curled and varied in thickness,and the lamellar structure was more uniform.Although some myelin were missing,the loosening degree was less than that of the Model group,and clear mitochondria were still visible,but the number was less than that of the Sham group,and the cell body was smaller.10.The morphology of dendrites and dendritic spines was observed by electron microscopy The spinal cord of each group was stained by Golgi,and the results showed that the spinal dendrites in the Sham group were complete in structure,regular in shape,long in length,densely covered with dendritic spines,which were distributed neatly,uniformly and densely,and in large number.In the Model group,the spinal dendrites were irregular in shape,partially broken and missing,short and slender in length,and the number of dendritic spines was small,sparsely distributed and disordered.Compared with the Model group,the morphology of spinal dendrites in DDD group,TGN-020 group and DDD+TGN-020 group were improved,with longer,more complete structure,thicker peripheral meridian,more number of dendritic spines,and more regular and orderly arrangement.Quantitative analysis of spinal dendritic length and dendritic spines in each group showed that compared with Sham group,dendritic length and number of dendritic spines in Model group were significantly decreased(P<0.05).Compared with Model group,the dendritic length and the number of dendritic spines in DDD group,TGN-020 group and DDD+TGN-020 group were significantly increased(P<0.05);There was no significant difference between DDD group,TGN-020 group and DDD+TGN-020 group(P>0.05).Part Ⅲ1.The primary AS was observed under the microscope The primary cells were cultured for 2 days.Microscopic results after extraction and purification showed that AS cells were scattered in irregular shape,some of which were stellate in shape,with large elliptical nuclei and abundant cytoplasm.From the cell body,long branched projections were sent out to the surrounding areas.After 4 days of primary cell culture,the cells proliferated rapidly and filled the bottom of the bottle with about 90%irregular shape,which could be subcultured.Through the passage to the third generation of AS,the cells grew in good condition and were suitable for subsequent experiments.2.AS identification By immunofluorescence staining,GFAP positive staining results showed that the cytoplasm of the cells was red,the nucleus was blue,and no positive staining was observed in the negative control.In conclusion,GFAP was specifically expressed and the extracted cells were AS.3.Safe intervention concentration of DDD drug-containing serum for AS,and optimal intervention time and concentration for RAS intervention The safe concentration range of DDD drug-containing serum for the intervention of AS was 0-15%.The optimal concentration of DDD drug-containing serum for the intervention of RAS was 10% and the optimal intervention time was 48 h.4.The influence of DDD containing serum on RAS activity MTT results showed that compared with CG group,RAS activity in other groups was decreased(P<0.05).Compared with MG,the activity of RAS in BSG group had no significant difference(P>0.05),and the activity of RAS in DDD,TGN-020 and DDD+TGN-020 groups was significantly decreased(P<0.05).There were no significant differences among DDD,TGN-020 and DDD+TGN-020 groups(P>0.05).5.The influence of DDD containing serum on RAS mobility24 h after injury,RAS was completely separated from contralateral cells in each group,and a blank scratch area was formed in the middle,and RAS was not seen or rare in the scratch area.After 48 hours,some cells began to extend synapses to the contralateral side,and some hypertrophic RAS appeared in the scar area.The cell cytoplasm was rich,the cell body was spherical,shiny,and the cell division was accelerated.After 72 hours,the scratch area shrank,part of RAS interwoven with contralateral RAS synapses,and the scratch began to heal.During the whole healing process,there was no significant difference in the scratch area ratio among all groups at 24h(P>0.05).From 48 h on,compared with MG group,the scratch area ratio of BSG was not significantly different,but the other groups were significantly larger (P < 0.05).There was no significant difference between DDD group,TGN-020 group and DDD+TGN-020 group(P>0.05).After 72 h,there was no significant difference in scratch area ratio between MG group and BSG group(P>0.05).Compared with MG group,the scratch area ratio of DDD group,TGN-020 group and DDD+TGN-020 group was significantly larger(P<0.05),but there was no significant difference between groups(P>0.05).The change of the scratch area ratio reflects the migration rate of RAS to the damaged area after injury.6.Influence of each group on the fluorescence expressions of AQP4 and GFAP in RAS AQP4,GFAP and nuclei in RAS were labeled by immunofluorescence staining.The staining results showed that AQP4+ cells were green,GFAP+cells were red,the nuclei were blue,and the fluorescent double-label cells were yellow and irregular dendritic.In CG group,only 1 to 2 AQP4+/GFAP+cells were found sporadically.There were more AQP4+/GFAP+ cells in MG group and BSG group,with higher brightness and severe cell swelling.There were fewer AQP4+/GFAP+ cells in the DDD group,TGN-020 group and DDD+TGN-020 group,with low brightness and mild cell swelling.AQP4+/GFAP+ cells were counted and the results showed that compared with CG,the proportion of positive cells in other groups was significantly increased(P<0.05).Compared with MG,the percentage of positive cells in DDD group,TGN-020 group and DDD+TGN-020 group was significantly decreased(P<0.05),except for BSG group(P>0.05).There was no significant difference in the percentage of positive cells in DDD group,TGN-020 group and DDD+TGN-020 group(P>0.05).7.Effects of each group on the protein expression levels of AQP4,GFAP,NGF,BDNF and GAP-43 in RAS The results showed that compared with CG group,the protein expression levels of AQP4 and GFAP in other groups were significantly increased(P<0.05),while the protein expression levels of NGF,BDNF and GAP-43 were significantly decreased(P < 0.05).Compared with MG group,the protein expression levels of AQP4 and GFAP in DDD group,TGN-020 group and DDD+TGN-020 group were significantly decreased(P < 0.05),and the protein expression levels of NGF,BDNF and GAP-43 were significantly increased(P<0.05),except for BSG group(P>0.05).Conclusion1.The self-made simple spinal cord impaction device can strike the spinal cord tissue vertically from the height of 20 mm and 40 mm.The animal model of spinal cord injury similar to that of NYU impaction device from the height of 12.5mm and 25 mm can be used for reference by researchers with high safety,good stability,simple operation and low price.2.Under the guidance of Tongfu-Zhuyu therapy,Didang decoction can effectively eliminate edema in SCI rats and promote neurological recovery.The specific mechanism of its action is as follows: inhibiting the activation of reactive astrocytes,reducing glial scar formation,increasing the expression of neurotrophic factor,nerve growth factor and cell membrane growth-related proteins,and promoting Neurological rehabilitation.3.DDD-containing serum can effectively reduce RAS edema,reduce RAS activation and slow down RAS migration,and promote the secretion of neurotrophic factors in RAS by inhibiting the expression of AQP4.