| At the clinical level, healing is defined as complete closing of the integument.For normal (nonchronic) healing of a primary intention wound, the healing process is complete in about 3 to 14 days. The sequence of events includes wound formation, inflammation, provisional matrix formation, collagen syntheses, epithelialization, neoangiogenesis and, finally, wound closure. Platelet-derived growth factor (PDGF) was identified as a serum growth factor for fibroblasts, smoothmusclecells (SMCs), and gliacells. In the progress of wound healing, PDGF can increase the collagen, hyaluronic acid synthesis and secreting. PDGF is a family of cationic homo- and heterodimers of disulfide-bonded A-/B-/C- and D- polypeptide chains.In addition, recombinant human PDGF-BB has been introduced in the clinic as a wound healing therapy. MMP-9 is a member of matrix metalloproteinases (MMPs) family. MMP-9 plays active role in wound healing being primarily responsible for the degradation of various extracellular matrix components over the course of wound healing. Compare to the pharmaceutical chemicals and the traditional Chinese medicine, genetically engineered drugs have many prombles in the pharmacology and the toxicology. Traditional Chinese medicines have a long history in the applicationin inflammations and immunological diseases. Because of the low incidence rate of adverse reaction, researching and developing the new traditional Chinese medicines for wound healing are very meaningful. Paeonol is the major active component of Paeonia suffruticosa and Nanchum paniculatum. Paeonol has some effects like anti-inflammation, anti-allergy.About the mechanism that how PDGF-BB increase wound healing, the present researches most focused on enhancing proliferation and migration of dermal fibroblasts. Our research focused on the PDGFR-β,PDGF-BB and MMP-9 at the same time and considerated the amount of receptors, degradation of ECM and secreting of inflammatory factors.Some researchs showed that an excess of TNF-αleads to damage of wound healing, resulting in a chronic wound. So in this research, we use TNF-αtreated the human dermal fibroblasts to modeling the internal environment of chronic wound. Inspected the changing of PDGFR-β,MMP-9 in the internal environment of chronic wound. Then treated the human dermal fibroblasts with PDGF-BB and TNF-αtogether, inspected the regulation of PDGF-BB on MMP-9 and TIMP-1. In the end, we treated the human dermal fibroblasts with PDGF-BB to inspect the regulation of PDGF-BB on the secretion of inflammatory factors. During this research, we also approached the effedts of paeonol.Through this research, we raised a possible mechanism of how PDGF-BB enchance wound healing. Approached the effedts of paeonol and offered a new idea in finding drugs which can increase wound healing.Partâ… : The effects of TNF-αand Pae on PDGFR-βin human dermal fibroblasts1. A method of culruring human dermal fibroblast was established. Investigated the effect of Pae on the survivability in human dermal fibroblasts.2. The results of RT-PCR showed that there was PDGFR-βmRNA existed in human dermal fibroblasts. 10μg·L-1TNF-αcan up-regulated the expression of PDGFR-βmRNA. Pae can inhibit this up-regulation caused by TNF-α.3. The results of flow cytometry showed that there was PDGFR-βexisted on the surface of cell membrane in human dermal fibroblasts. The base of M1 is 28.65±0.06%. 10μg·L-1TNF-αcan up-regulated the M1 of PDGFR-βto 41.13±5.30%. Treated the human dermal fibroblasts with 10mg·L-1 Pae alone can up-regulated the M1 of PDGFR-βto 37.35±0.31%. Co-incubation 10μg·L-1TNF-αand Pae(10,50,100 mg·L-1), the M1 are 41.10±1.29%, 39.96±0.91%, 33.32±0.95% , respectively. Shows no difference in statistics.The results above indicated that in the the internal environment of chronic wound modeled by TNF-α, the mRNA and Ml of PDGFR-βwere up- regulated. The increased number of receptors can enhance the effects of PDGF-BB, promote the proliferation and migration of fibroblasts better.Partâ…¡: The effects of TNF-αand Pae on MMP-9 in human dermal fibroblasts1. The results of RT-PCR showed that there is rarely MMP-9 mRNA expressed in human dermal fibroblasts. 10μg·L-1TNF-αcan up-regulated the expression of MMP-9 mRNA. Pae can inhibit this up-regulation of MMP-9 mRNA caused by TNF-α.2. The results of Western blotting showed that there is rarely MMP-9 expressed in human dermal fibroblasts. 10μg·L-1TNF-αcan up-regulated the expression of MMP-9. Co-incubation 10μg·L-1TNF-αand Pae(10,50,100 mg·L-10, Pae can inhibit this up-regulation of MMP-9 to 8.77%, 14.04% and 10.53% caused by TNF-α, respec- tively.The results above indicated that in the the internal environment of chronic wound modeled by TNF-α, the mRNA and molecule of MMP-9 are up-regulated. Over-expressed MMP-9 can cause the ECM over-degration and delay the healing of wound.Partâ…¢: The effects of PDGF-BB on MMP-9 in human dermal fibroblasts1. The results of Western blotting showed that there is rarely MMP-9 expressed in human dermal fibroblasts. 10μg·L-1TNF-αcan up-regulated the expression of MMP-9. Co-incubation 10μg . L-1TNF-αand PDGF-BB(2,5,10μg·L-1), PDGF-BB can inhibit this up-regulation of MMP-9. 10μg·L-1PDGF-BB can down-regulated 44.68% of the MMP-9 expression. Co-incubation 10μg·L-1TNF-αand100mg·L-1 Pae can also down-regulated 48.94% of the MMP-9 expression.2. The results of ELISA showed that there is rarely MMP-9 screting from human demal fibroblasts, the base line is 2.328±0.005 pg·mL-1. 10μg . L-1TNF-αcan up-regulated the screting of MMP-9 to 7.371±2.179 pg·mL-1. Co-incubation 10μg·L-1TNF-αand Pae(10,50,100 mg·L-1), Pae can inhibit this increased screting of MMP-9. 100 mg·L-1Pae can down-regulate the screting of MMP-9 to 2.325±0.001 which is very close to the baseline.3. The results of ELISA showed that the base line of MMP-9 screting is 12.952±0.3161pg·mL-1 from human demal fibroblasts. Treated with 10μg·L-1TNF-αfor 24h, the screting is up-regulated 35.01%. Co-incubation 10μg·L-1TNF-αand PDGF-BB(2,5,10μg·L-1), PDGF-BB can inhibit this increased screting of TIMP-1. 100 mg·L-1Pae can down-regulate the increase of MMP-9 screting caused by 10μg .L-1TNF-αto 12.321±1.093pg·mL-1.4. By analysing the MMP-9/TIMP-1, we can know that the baseline of MMP-9/TIMP-1 in human dermal fibroblasts is 0.180. Treated with 10μg·L-1TNF-αfor 24h, the ratio rasied up to 0.434. Co-incubation 10μg·L-1TNF-αand PDGF-BB(2,5,10μg . L-1), PDGF-BB can inhibit the ratio of MMP-9/TIMP-1 which means that the balance is recovered. 10 mg·L-1Pae has the similar effects with PDGF-BB.The results above indicated that in the the internal environment of chronic wound modeled by TNF-α, PDGF-BB can down-regulate the expression and screting of MMP-9 and recover the MMP-9/TIMP-1 balance which may be the possible mechanism of enhance wound-healing. 100 mg·L-1 Pae has the similar effects with PDGF-BB which suggests the potential of curing of chonic wound healing.Partâ…£: The effects of PDGF-BB and Pae on screting of cytokine in human dermal fibroblasts1. The effects of PDGF-BB and Pae on the screting of IFN-γwere measured by ELISA. The results showed that the baseline of screting of IFN-γis 28.704±4.4E-15 pg·mL-1. Treated the human dermal fibroblasts with 10μg·L-1PDGF-BB, the screting is down-regulated 12.68%. Co-incubation 10μg·L-1 PDGF-BB and Pae(10,50,100 mg·L-1), compare to the control, 50mg·L-1Pae can increased the screting of IFN-γ.2. The effects of PDGF-BB and Pae on the screting of IL-6 were measured by ELISA. The results showed that the baseline of screting of IL-6 is 193.965±22.634 pg·mL-1. Treated the human dermal fibroblasts with 10μg·L-1PDGF-BB, the screting is up-regulated to 238.513±11.568 pg·mL-1. Co-incubation 10μg·L-1 PDGF-BB and Pae(10,50,100 mg·L-1), the screting of IL-6 are all down- regulated.3. The effects of PDGF-BB and Pae on the screting of IL-8 were measured by ELISA. The results showed that the baseline of screting of IL-8 is 755.042±34.113 pg·mL-1. Treated the human dermal fibroblasts with 10μg·L-1PDGF-BB, the screting is up-regulated to 1025.120±71.775 pg·mL-1. Co-incubation 10μg·L-1 PDGF-BB and Pae(10,50,100 mg·L-1), compare to the control, the screting of IL-8 are all down-regulated. 10 mg·L-1Pae down- regulated most, 50,100 mg·L-1Pae down- regulated the screting of IL-8 20.49% and 22.73%, respectively. The results above indicated that in the the internal environment of chronic wound modeled by TNF-α, PDGF-BB can increase the IL-6, IL-8 and decrease the IFN-γ. So, enhancing inflammatory reaction may be another possible mechanism of PDGF-BB in enhancing wound-healing. Pae can down- regulated the increase of IL-6 and IL-8 by PDGF-BB showed excellent anti- inflammatory effects. If we treated the patients with Pae at the right time, the healing may be leaded to a scarless wound healing.
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