Epidural fibrosis is a deformed healing of the incision site after laminectomy. In this process, fibroblasts accumulate at the incision after a large number of proliferation. As a pathological process, epidural fibrosis can cause a large amount of fibrotic tissue to form in the surgical site after laminectomy, which seriously affects the surgical effect of patients. The mechanism of the proliferation of fibroblasts is relatively complicated, and there are still many related problems that need to be solved. However, it is generally recognized that excessive proliferation of fibroblasts is the main factor causing tissue fibrosis[21–23].
TGF-β1 has extensive biological functions, including regulating cell growth, differentiation, migration, matrix formation, and damage repair[24–26]. It is the most in-depth and most powerful profibrotic cytokine currently studied[27–29]. TGF-β1 is a powerful chemokine for monocytes and fibroblasts. As well as, it can increase the proliferation ability of fibroblasts, promote their secretion of extracellular matrix such as collagen and fibronectin. TGF-β1 can induce fibroblasts phenotype Transformation to myofibroblasts phenotype, characterized by high synthesis of α-SMA, causing matrix contraction and participating in accelerating the extracellular matrix deposition. It also has the function of repressing extracellular matrix degradation and promoting apoptosis of epithelial cells. At the same time, it is an important cell that induces epithelial-mesenchymal transition, making epithelial cells lose their polarity, adhesion molecules such as cadherin, and obtain characteristics of mesenchymal cells, such as the synthesis of fibronectin, α-SMA, and exhibit migration. It can be seen that it has a significantly impacts on the mechanism of epidural fibrosis formation.
The Smad protein is the main signal transduction factor of TGF-β1, which transfers the signal of TGF-β1 from the cell to the nucleus regulates target gene transcription. Co-regulates the transcription of target genes with other transcription factors. Many articles indicate that repress the TGF-β1 channel can inhibit fibroblasts functions[37–39].
MMF is a derivative of mycophenolic acid (MPA), and it is a new immunosuppressant. Rapid hydrolysis of MMF after oral administration into the active metabolite MPA in the body. MPA inhibits the synthesis of guanine nucleotides by repressing the key rate-limiting enzyme inosine monophosphate dehydrogenase (IMPDH), a new channel for the synthesis of purine nucleotide. Due to its specificity, MMF has recently been used in the treatment of proliferative fibrotic diseases and has achieved good results. Badid et al.found that 72 hours after MMF treatment, MMF can dose-dependently inhibit rat fibroblasts proliferation, reduce infiltration of interstitial myofibroblasts and deposition of type III collagen. Liu et al. found that MMF improves renal function and reduces renal interstitial fibrosis in a rat renal interstitial fibrosis model with 5/6 resection of the kidney. Nina and other studies found that MMF can inhibit the expression of collagen genes, the contraction of extracellular matrix, and the migration of fibroblasts suggest that it may have an anti-fibrotic effect. Our experimental results also proved the anti-fibrosis effect of MMF. Dubus et al. found that MMF can reduce TGF-β1-induced human epithelial cell proliferation and extracellular matrix production (such as type I collagen and laminin deposition). It can be seen that MMF can not only inhibit lymphocyte proliferation and recruitment, but also have a direct anti-fibrosis effect, but there is no related research on epidural fibrosis. We speculate that MMF can terminate the signal transduction of TGF-β1-related channel, thereby inhibiting the proliferation, migration and differentiation of fibroblasts. Our study showed that MMF could also inhibit the differentiation of fibroblasts, which was barely involved in the above studies, and our study on cell differentiation helped deepen the understanding of the mechanism of MMF anti-fibrosis.
Therefore, we speculated that MMF may repress the functions of fibroblasts by regulating the TGF-β1 channel. In this research, we used Western blot to research fluctuation in related protein synthesis. The results showed that the synthesis quantities of TGF-β1, p-Smad2, p-Smad3, PCNA and Cyclin D1 decreased after MMF treatment. These results indicate that MMF can regulate TGF-β1 channel and its channel proteins to repress fibroblasts proliferation.
In vitro experiments, we conducted a number of cell experiments to investigate whether MMF can inhibit the proliferation, migration, and differentiation of fibroblasts. Our results indicate that MMF can play these roles. At the same time, we also found that MMF significantly repressed the TGF-β1 / Smad2/3 axis. Therefore, we used a potent activator, TGF-β1, to activate the channel. According to the results of Western blot analysis, we found that TGF-β1 effectively activated the TGF-β1 / Smad2/3 axis. The activated channel attenuated impacts of MMF on the proliferation, migration, and differentiation of fibroblasts.
In vivo experiments, we established a laminectomy model in rats. After laminectomy, different concentrations of MMF were applied locally to rat incisions. The selection of the MMF concentrations was based on previous studies in rats. H&E-staining histological analysis of epidural fibrotic tissues demonstrated that fibrosis in the fibrotic tissues from the surgical areas in the MMF treatment group was significantly weaker, and the number of fibroblasts was significantly reduced. We used Masson's trichrome staining to evaluate collagen content in fibrotic tissues. Our results showed that MMF can significantly inhibit collagen content. Further research on histochemical staining demonstrated that the synthesis of PCNA, tubulin and α-SMA in fibrotic tissues in the group treat with MMF was obviously less than in the saline group. These studies indicate that MMF could reduce epidural fibrosis caused by operation.
In conclusion, based on above results, we can preliminary explain the role of MMF on inhibiting epidural fibrosis. In short, when applying MMF to the laminectomy site, the TGF-β1 / Smad2/3 axis in fibroblasts is repressed, and finally the proliferation, migration and differentiation of fibroblasts are inhibited, thereby reducing the fiber formation.
The mechanism of epidural fibrosis formation is intricacy, there are still some limitations in our research. Firstly, MMF was administered topically. Although all animals survived until the study finished, a few side effects may happen on other regions. Secondly, we only studied the effect of TGF-β1 / Smad2/3 axis in inducing fibroblasts proliferation, migration, and differentiation, without further exploring MMF may affect fibroblasts proliferation through other signaling pathways. These are the shortcomings of this research, and we will start to conduct more in-depth research in these areas in the future.