Experimental And Clinical Studies Of Application Timing Of Shujin Fumigation After Fracture Surgery

ObjectiveShujin fumigation is a method of treatment more applications on the orthopedic clinic, which has Shujin, stasis swelling effect and can promote the recovery of limb function. However, after some fractures were treated by traditional Chinese medicine which was Shujin fumigation in clinical practice, we found that the emergence of local skin was redness, soft tissue was swelling and pain, which even cause severe sclerosis and ossification of soft tissue. But the method of delayed patients was less these reactions. Why is there such adverse reactions? If is there a correlation that application timing of herbal fumigation were keeping with the incidence of adverse reactions? When is an appropriate application of Chinese medicine fumigation?This paper aims to explore reasons of soft tissue injury after shujin fumigation and its mechanism of adverse reactions in damage symptoms, histopathology, cytokines, which we adopt histological, molecular biology techniques. At different points, the method of ELISA checks the content of IL-1β and IL-6after meridians fumigation intervention. The methods of immunohistochemistry, roal-time PCR and Western-blot detect the combination of soft tissue injury in PDGF and TGF-β protein expression. To provide a theoretical basis, we study the rational application of herbal fumigation intervention. In clinical research, the elbow fracture is represented. At different points, we use meridians fumigation intervention after trauma fracture surgery to explore herbal fumigation applications in pain, swelling, elbow ROM, skin reactions conditions, safety and other aspects of Mayo elbow performance score. Methods1.The first part of experimental studyThe rats of of healthy and adult have sixty. They are the weight of220±20g, and male and female in each half. The rats were adapted to feed after a week, and were made model. After modeling, drug design standards were in accordance with NDST, the rats were randomly divided into four groups:normal rat,model group, saline group, shujinxi granule group,the group has15rats.The rats modeling (wound has healed) started interventions after two weeks. The shujinxi group use the drug of shujinxi granule to wash fumigation limb. The saline group use0.9%sodium to wash fumigation limb. The model group did not do not use any intervention. The normal group has no treatment. Shujinxi group and saline group fumigate (temperature controlled at40-45℃) twice in every day, which fumigate30minutes. After seven, fourteen and twenty-one days, the two groups were draw materials. The other two group were draw materials at the same time. All groups were sacrificed every time. The observation and testing indicators have injury symptoms index, enzyme-linked immunosorbent assay detection (ELISA) of IL-1β and IL-6, HE staining pathological morphology, immunohistochemistry, real-time PCR and Western-blot detection methods of combining the soft tissue injury and TGF-β and PDGF protein expression. Statistical methods use SPSS16.0statistical software, the mean comparing of groups use ANOVA. If the homogeneity of two growps are variance, we use the least significant difference method (LSD). If heterogeneity are variance between groups, we use Dnnett method.2. The second part of the experimental studyThe rats of of healthy and adult have sixty. They are the weight of220±20g, and male and female in each half. The modeling is above. After modeling, drug design standards are in accordance with NDST, the animals were randomly divided into A, B, C and D groups. Every group has15rats. A group begin to use shujinxi fumigation in the second week after modeling (wound has healed), B group is in the third week after modeling, C group is in the fourth week after modeling, and D group have not any interventions after modeling. The rats of A, B, C group were performed fumigation twice every day (temperature controlled at40-45℃), which fumigate30minutes. The each observation period is three weeks. Every week, five rats were killed to drown materials per group. The observation and testing indicators have injury symptoms index, enzyme-linked immunosorbent assay detection (ELISA) of IL-1β and IL-6, HE staining pathological morphology, immunohistochemistry, real-time PCR and Western-blot detection methods of TGF-β and PDGF protein expression. Statistical methods above.3.Clinical ResearchThis study involved the elbow fracture. The patients are from orthopedic inpatients of Foshan Hospital of Chinese Medicine. The patients are120cases in all. The cases were collected from June2012to December2013. The cases were randomly divided into A, B, C and D groups in random order:A group of30patients,17males and13females, mean age (33.03±9.53); B group of30cases,18males and12females, mean age (34.90±10.20); C group of30cases,15males and15females, mean age (34.66±10.89); D group of30patients,16males and14females, mean age (32.50±9.53).The above cases were fresh fractures. The general information of four groups were compared. The difference was not statistically significant (P>0.05). The observation and testing indicators have pain, swelling, elbow ROM evaluation, skin reactions, safety and other aspects of Mayo elbow performance score. A group are intervented with shujinxi fumigation in the first three weeks after surgery (wound has healed). Group B starts at4weeks after surgery. Group C begins after the first five weeks. Group D have no any intervention (control group). Statistical methods use SPSS16.0statistical software, the mean comparing of groups use ANOVA. If the homogeneity of two growps are variance, we use the least significant difference method (LSD). If heterogeneity are variance between groups, we use Dnnett method. If the date does not meet the normal distribution, the measurement data will use non-parametric tests.Results1. The first part of the experimental resultsThe effect of intervention measures on injury symptom index:In7days after the intervention, normal saline group and tingluoxi group score values are basically identical and score values of two group were higher than that of model group. In the first14days and21days after intervention, model group and normal saline group score values both have a downward trend and tingluoxi group score value also has a downward trend, but the score values in different periods were higher than that of model group and normal saline group. Intervention effects on Serum IL-6levels:At different times after the intervention, the levels of serum IL-1β saline group and tingluoxi group are both higher than that of model group rats and normal group, and tingluoxi group is higher than that of normal saline group. HE staining pathology morphological observation:It is obvious in normal rats group that muscle fiber distribution is consistent and nuclear distribution is evener, located in the peripheral portion of the cytoplasm; In different period, It can be seen in the model group that parts of the muscle fibers loosely arranged, with a small amount of inflammatory cells presented; In the7days and14days after intervention in the saline group inflammatory cells can be seen scattered among muscle fibers. In the21days after the intervention, the connective tissue hyperplasia is obvious and there are capillary hyperplasia and more inflammatory cells; tingluoxi group in7days after the intervention, inflammation cells subcutaneous tissue infiltrated, scattered hemorrhage.14days after intervention, scattered inflammatory cells among muscles were more, in the21st day after the intervention, connective tissue hyperplasia. There were obviously visible fiber mother cell and a lot of inflammatory cells。 Interventions for the effects on PDGF:Immunohistochemical detection showed, In different periods after the intervention, PDGF protein expression in the soft tissues of both tingluoxi group and saline group were higher than that of model group and normal rats group, and tingluoxi group was higher than normal saline group。Real-time fluorescent quantitative PCR, Western blot detection results showed consistent with immunohistochemical results.The influence of intervention measures on TGF-β:Immunohistochemical detection showed, In7and14days after intervention, TGF-β protein expression of soft tissue in both tingluoxi group and saline group was significantly higher than that in model group and normal rats group and tingluoxi group is higher than normal saline group。In21days after the intervention, TGF-β protein expression of soft tissue injury in tingluoxi group were higher than that in saline group, model group and normal rats group respectively. Real-time fluorescent quantitative PCR and Western blot detection results showed consistent with immunohistochemical results.2. The second part of the experimental resultsThe effect of intervention measures on injury symptom index:In different periods after the intervention, group A score value is higher than that of group B, group C and group D. Intervention effects on serum IL-1β level: After the intervention in different periods, serum IL-1β level of group A was significantly higher than that in group B, group C and group D and serum IL-1β level in group B was significantly higher than that of group C。Intervention effects on serum content of IL-6:In different periods after the intervention, IL-6level in serum of group A is higher than that in group B, group C and group D and serum IL-6level in group B is higher than that of group C. HE staining pathology morphological observation:With ongoing intervention, inflammatory cells among muscle in Group A gradually increased and there were still a large number of inflammatory cells and connective tissue hyperplasia even at the last stage. The number of inflammatory cells among muscle fibers gradually reduced with the intervention on group B; With ongoing intervention on group C, the number of inflammatory cells among muscle fibers also gradually reduced, relatively less than that of group B however; Fumigation interventions were not used in group D. In different periods of intervention, parts of the muscle fibers loosely arranged and there were only a small amount of inflammatory cells.Interventions for the effects on PDGF:Immunohistochemical detection results showed that in different periods, after intervention the grey value of PDGF of soft tissue injury in the group A was higher than that of group B, group C and group D and the gray value of of PDGF of soft tissue injury in group B was higher than that in group C. Real-time fluorescent quantitative PCR and Western blot detection results showed consistent with immunohistochemical results.The influence of intervention measures on TGF-β:Immunohistochemical detection results showed that in different periods after intervention, the amount of soft tissue TGF-β protein expression in group A were higher than that of group B, group C and group D and the soft tissue TGF-β protein expression in group B was higher than that of group C. Real-time fluorescent quantitative PCR and Western blot detection results showed consistent with immunohistochemical results.3. Result of clinic studyIn different period of intervention, the pain scores of group A is the highest, followed by group B and group C.Swelling:In different period of intervention, the most severe swelling showed in group A, followed by group B and group C. Elbow ROM meter (including buckling, back stretch, pronation and supination):In different periods after the intervention, elbow range of group A is minimum, elbow motion of group B is bigger, and elbow range of group C is the largest.Mayo elbow function evaluation.-In different periods after the intervention, group A got worst elbow joint function recovery, elbow joint functional recovery is better than that of group A and group B C group was better than that of group A and group B. In particular, at the time of the last follow-up, Mayo elbow function score of group C was the best, group B was better, and group A was goog?Skin reaction:Erythema was found only in one patient as external use of fumigation for high temperature, nothing to do with drugs.Safety evaluation:During the test, each group patients were well tolerated, with no adverse reaction, the score was0.Conelusion1. In the stage of cell differentiation and proliferation of wound repair, we use different intervention measures of saline and Shujinxi granule fumigation, and found that they improved the content of IL-1β and IL-6in serum. Their overexpression and secretion can cause local inflammatory reactions, thereby affect the wound healing process. HE staining can be further verified from pathologic morphology. Fumigation intervention can cause local inflammatory reaction, and cause hypertrophic scar. Because the Shujinxi fumigation were more serious than saline in the inflammatory factors, symptom index and pathology, so we think Shujinxi fumigation caused inflammatory response more than saline.2. After using fumigation intervention, they caused excessive secretion or expression in PDGF and TGF-β,and can lead to hypertrophic scar or keloid. Shujinxi fumigation is the most serious, so that the improper fumigation intervention will have an adverse effect on wound healing.3. Shujinxi fumigating intervention can cause inflammatory reaction at different time, so the first four weeks is not suitable for use of Shujinxi granule for fumigation intervention.4. The first four weeks after trauma is not suitable for using Shujinxi fumigation intervention, otherwise it may cause local hypertrophic scar or fibrosis, and affect the wound repair process.5. We found that the patient created pain, swelling and poor recovery of elbow joint function from the conclusion of the experimental research results combining with clinical results. These may relate with much secretion and abnormal expression of IL-1βL-6, PDGF, TGF-β. These may cause the aggravating, the local inflammatory reaction excessive fibrosis and hypertrophic scar. Then these can cause tendons and muscles fibrous adhesions, granulation tissue hyperplasia, which influence joint activities and functions. In the clinical stage, fumigation intervention is not appropriate in the cell proliferation and differentiation (the fifth weeks after fracture operation before).