The Role And Mechanism Of Tongxinluo Joint PBSCT In The Treatment Of DFU In Rats

Objective: This study observed the effects of Tongxinluo joint PBSCTon general status, local ulcer recovery, microvessel density, inflammatory andoxidation-related factors level, expression of angiogenesis-related factors inlocal tissue of DFU(diabetic foot ulcer) rats. The potential mechanism of thetreatment method was explored from promoting ulcer healing and theangiogenesis and inhibitting oxidative damage and improving localmicroenvironment.Methods:1The treatment effects of TXL joint PBSCT on DFU ratsThe diabetic animal model was developed with High-fat diet combined45mg/kg streptozotocin (STZ) intraperitoneally injection, land iquid nitrogenfreezing swab of diabetic rats was used to establish DFU rats model. Aftermodel established,the rats were randomly divided into follow group:modelgroup rats(Model) were given an equal volume of0.5%CMC-Na solutiongavage and equal volume of sterile injectable medium RPM1640;TXL grouprats (TXL) were given TXL0.8g/kg body weight and an equal volume ofsterile injectable medium RPM1640;stem cell group rats (Stem) were given anequal volume of0.5%CMC-Na solution gavage and stem cell transplantation;TXL+stem cell group rats (T+S) were given tongxinluo0.8g/kg body weightand stem cell transplantation.resulting in control group rats (Control) the samebatch set foot ulcers in rats received the same volume of0.5%CMC-Nasolution gavage and equal volume of sterile RPM1640medium injection.Themethod of Stem cell transplantation: At the edge of foot ulcers1mmequidistant four injection sites of about0.1ml of cell suspension, six points incalf muscles,0.05ml cell suspension was injeced in each point. General stateof the rats, fasting blood glucose, body weight, blood lipids, serum insulinlevels, foot ulcer healing, HE staining and frozen section histological changes were observed.2The influence of angiogenic in DFU rats trezted by TXL joint PBSCTIt was the same with part1that experimental animals modeling,grouping,administration and derived method.The ELISA was adopted to detect VEGF.The mRNA expression of VEGF were detected by Real-time PCR.Local tissueCD34expression in Diabetic foot ulcers rats were detected byimmunohistochemical technology.Western-blot was used to detect CD34,CD44, CD105expression of ulcers local tissue.3The effects of TXL joint PBSCT on oxidative damage and cell adhesionmolecules in diabetic foot ulcers ratsIt was the same with part1that experimental animals modeling, grouping,administration and derived method.We detected the levels of serum ROS,AGE, RAGE, advanced glycation end product receptor (RAGE) mRNAexpression by real-time PCR, and ICAM-1, VCAM-1, Nrf-2, and HO-1protein expression of assay ulcers local organizations by western-blot.Results:1The treatment effects of TXL joint PBSCT on DFU ratsThe results rats fasting body weight and blood glucose: After7days and28days of treatment, compared with the control group, blood sugar increasedand weight loss in model group (P<0.05or P<0.01); Compared with themodel group, blood sugar decreased and weight gain in T+S group (P<0.05);compared with stem group, blood sugar decreased and weight gain in T+Sgroup (P<0.05).Lipid results of rats: After7days and28days of treatment, comparedwith control group, serum TG, CHOL, LDL increased, HDL decreased inmodel group (P<0.01);compared with the model group, serum TG, CHOL,LDL decreased, HDL increased in TXL group and T+S group; compared withstem group, serum TG, CHOL, LDL decreased, HDL increased in T+S group(P<0.05or P<0.01).The results of rats serum insulin: After7days and28days of treatment,compared with control group, serum insulin decreased in model group (P<0.01); compared with the model group, serum insulin increased in TXLgroup and T+S group(P<0.05or P<0.01); compared with Stem group, seruminsulin increased in TXL group and T+S group(P<0.05or P<0.01).The rats ulcer healing observed: Compared with the control group, theulcer healing was poor in model group; compared with the model group, theulcer healing was better in treatment groups, T+S group was the best. HEstaining pathology observed, compared with the control group, there was lessgranulation tissue and more inflammatory cell in model group; compared withthe model group, there was more granulation tissue and fewer inflammatorycells in all treatment groups. After stem cell transplantation7days, under afluorescence microscope visible frozen section of Stem group and T+S groupwithin has a large blue fluorescence gathered, T+S group is higher than theStem group.2The influence of angiogenic in DFU rats trezted by TXL joint PBSCTThe serum VEGF levels in each group of rat: After7days and28days oftreatment, compared with the control group, VEGF levels reduced in modelgroup (P<0.01); compared with the model group, serum VEGF levels wereincreased in the treatment groups (P<0.05or P<0.01);compared with Stemgroup VEGF levels increased in TXL group and T+S group (P<0.05orP<0.01).VEGFmRNA expression in local tissue ulceration: After7days and28days of treatment, compared with the control group, VEGFmRNAexpression decreased in model group (P<0.01); compared with the modelgroup, VEGFmRNA expression increased in TXL group and TXL+stem cells(P<0.05or P<0.01); compared with stem cell group, VEGFmRNA expressionincreased in TXL group and T+S group(P<0.01).CD34expression in local tissue ulceration of rats: CD34protein positiveexpression is tan dyeing, mainly in the diabetic foot ulcer local tissuefreshmen expressed in vascular endothelial cells. Compared with the controlgroup, CD34expression was low of local tissue ulceration in model group;compared with the model group, CD34expression was higher of local tissue ulceration in the treatment groups, T+S group was higher than the othergroups.Western-blot detection of CD105, CD34, CD44protein expression inulcers local tissue:After7d、28d of treatment, compared with the control group,model group, CD34, CD44, CD105expression decreased (P<0.01); comparedwith the model group, the treatment groups ulcer local tissue CD34, CD44,CD105expression was increased (P<0.05or P<0.01); compared with the stemcell group, T+S group CD34, CD44, CD105expression were increased(P<0.05or P<0.01).3The effects of TXL joint PBSCT on oxidative damage and cell adhesionmolecules in diabetic foot ulcers ratsAfter7days and28days of treatment, compared with the control group,model group serum ROS, AGE, RAGE increased in model group(P<0.01);compared with the model group, levels of ROS, AGE, RAGE content inserum decreased in TXL group and T+S group (P<0.05or P<0.01); comparedwith Stem group, serum ROS, AGE, RAGE levels decreased in T+Sgroup(P<0.05or P<0.01).ICAM-1, VCAM-1protein content ulcers local tissus of rats: After7daysand28days of treatment, compared with control group, ICAM-1, VCAM-1expression increased of local tissue ulceration in the model group(P<0.01);compared with the model group, ulcer local tissue ICAM-1, VCAM-1expression decreased in treatment groups(P<0.05or P<0.01); compared withStem group, ulcers local tissue ICAM-1, VCAM-1expression was reduced inTXL group and T+S group (P<0.05or P<0.01).Local tissue ulceration Nrf-2, HO-1protein levels: After7days and28days of treatment, compared with the model group, local tissue Nrf-2, HO-1expression were increased in the treatment groups(P<0.05or P<0.01);Compared with the Stem group, tissue ulceration Nrf-2, HO-1expressionincreased in TXL group and T+S group(P<0.05or P<0.01).Local tissue ulceration RAGEmRNA expression of rats: After7days and28days of treatment, compared with the control group, RAGEmRNA expression increased in model group(P<0.01); compared with the modelgroup, RAGEmRNA expression in local tissue reduce in TXL and T+S group(P<0.05or P<0.01);compared with stem group, RAGEmRNA expressingdecreased in TXL group and T+S group(P<0.01).Conclusion:1TXL joint PBSCT therapy can significantly improve the general state ofdiabetic foot ulcers in rats, increased serum insulin levels, and promote thegrowth of granulation tissue, promote ulcer healing.2TXL joint PBSCT can promote ulcer serum VEGF expression and localorganizations to promote stem cell differentiation and promote angiogenesis.3Meridians drug combination AGE inhibition of peripheral blood stemcell transplantation and its receptor, inhibition of oxidative stress and adhesionmolecule expression.Meridians drug combination peripheral blood stem cell transplantationmay relieve ulcer from local tissue inflammation oxidative damage, promotestem cell differentiation, improve local micro-environment, promoteangiogenesis plays for the treatment of diabetic foot ulcers.