盐酸塔斯品碱对创面愈合的促进作用及其机理研究/Effect of taspine hydrochloride on skin wound healing in
塔斯品碱（Taspine，TA）是红毛七中提取得到的生物碱，我们通过细胞膜色谱法筛选发现：塔斯品碱、盐酸塔斯品碱（Taspine hydrochloide，TA/HCl）等与血管内皮细胞膜及成纤维细胞膜有很强的亲合特性。同时有文献报道，从南美植物巴豆属Croton lechleri中提取的塔斯品碱具有抗炎和促进创面愈合作用，但其作用机制尚不明确。我们实验发现塔斯品碱具有促创面愈合作用，但塔斯品碱在水中溶解度很小，为了改善其溶解性，将其制成盐酸盐。本研究在证明盐酸塔斯品碱对大鼠皮肤缺损创面有明显的促愈合作用的基础上，对其促进愈合的机理进行了探讨。
与模型组比较，创伤后3～7天，TA/HCl 2 mg•mL-1组肉芽组织中蛋白质的含量均明显升高（p<0.05）。创伤后3～14天，TA/HCl各给药组肉芽组织中Hyp含量明显升高（p<0.05或p<0.01）。
采用经典的巨噬细胞吞噬中性红实验，观察TA/HCl对巨噬细胞形态及吞噬率的影响。结果显示：TA/HCl在一定浓度范围（0.15～0.625 µg•mL-1）内，对巨噬细胞形态及吞噬率无明显影响；当浓度大于1.25 µg•mL-1时，对巨噬细胞的活性有抑制作用。
体外培养成纤维细胞，观察 TA/HCl对细胞增殖的影响。结果：TA/HCl浓度为0.01～0.50 μg•mL-1时，对成纤维细胞既无毒性作用，也无促增殖作用。
采用免疫组化和原位杂交的方法，观察创面组织中EGF、TGFβl / TGFβl R的蛋白水平及TGFβ1m RNA表达的变化。结果显示：创伤后3～21d，给药组伤口肉芽组织EGF、TGFβl / TGFβl R、TGFβ1m RNA表达有明显时相性。创伤后3d见阳性表达，7d表达达高峰，随后逐渐减弱，21d为弱表达。与模型组比较，TA/HCl 2 mg•mL-1组和bFGF对照组各时相点表达量比较，差异有显著性（p<0.05；p<0.01）。
采用免疫组化的方法，观察创面组织中纤维连接蛋白、层粘连蛋白及Ⅰ、Ⅲ型胶原水平的变化。结果显示：创伤后3～21d，纤维连接蛋白、层粘连蛋白及Ⅰ、Ⅲ型胶原在组织中的表达部位和水平不同，但TA/HCl 2mg•mL-1组和bFGF对照组对纤维连接蛋白、层粘连蛋白及Ⅰ型胶原的形成有明显的调节作用， 对Ⅲ型胶原影响不明显。
采用免疫组化和原位杂交的方法，观察创面组织中MMP1、TIMP1蛋白水平和TIMP1mRNA，MMP9mRNA表达的变化。结果显示：在整个修复过程中，给药组伤口肉芽组织MMP1、TIMP1、TIMP1mRNA，MMP9mRNA在组织中的表达部位和水平不同，表达有明显时相性。与模型组相比， TA/HCl 2mg•mL-1组和bFGF对照组阳性表达与模型组相比有显著差异。
Wound healing is a localized biological event that progresses through three general phases of inflammation: wound cell migration, mitosis, and extracellular matrix production and remodeling. The process of wound healing begins immediately following surface lesions or when skin proteins become exposed to radiation, chemical damage or extreme temperatures. Traditional treatments of wound are sutura, debridement, skin transplantation and antiseptics. Several topical wound medicants necessary for adequate wound healing. Optimal wound healing is a highly regulated sequence dependent on tissue cytokines or growth factors produced and released by platelets, polymorphonuclear leukocytes, monocytes, and macrophages in the local wound area. Traditional Chinese medicine had made important contribution to generation and health of the Chinese nation. Taspine hydrochloride had a good clinical prospect and deserved to be further as an accelerator for wound repair.
Taspine is an alkaloid extracted from Leontice robustum (root and rhizome of leontice robustum (Maxim.) Diels) in our laboratory. We screened by cell membrane chromatography (CMC) technology, and found that there were affinity interaction between taspine/ taspine hydrochloride and receptors in the membrane of vascular endothelial cell (ECV304) and fibroblast, which indicated there were bioactive components for wounding healing. Taspine extracted from trees of Croton of the western Amazon region was reported that taspine has anti-inflammatory and activities of promotion of wound healing, but its mechanism of action has not been elucidated completely. We also found the effect of taspine as an enhancer of wound healing. For improving its deliquescence, it was prepared as a water-soluble salt. The present study was conducted to evaluate the effect and mechanism of taspine hydrochloride on wound healing. The main results were as follows:
1. The retention features of tapine and tapine hyrochloride on the cell membrane chromatography (CMC) model
Epithelial cell and fibroblast (FB) cell membrane chromatography model were founded systematically based on the theories of drug-receptor interaction and primary CMC model. By the bioaffinity feature between drug and receptor (or protein), the CMC model could show drug’s bioactivity on certain tissue. Taking epidermal growth factor and basic fibroblast growth factor as model drugs, was studied systematically and found it showing specific binding character between drug and cell membrane. The results showed that the different polarity sites of taspine and taspine hydrochloride had obvious retention features on FB-CMC model, which indicated that there were bioactive components acting on Fibroblast system, and hadn’t retention features on FB-CMC model.
2. Effect of taspine hydrochloride on skin wound healing in rats
Bilateral round wounds were made on the backs of SD rats. The effect of taspine hydrochloride on the skin wound was evaluated through determining closure time and contracting ability of the skin wound, observing histopathological characteristics and measuring contents of hydroxyproline and protein in the wound tissue. The results showed that: The closure time of the skin wounds was shorter significantly in the taspine hydrochloride-treated groups than that in the model group. The percentages of wound contraction were higher in the taspine hydrochloride-treated groups than that in the dimethyl sulfoxide (DMSO) control group (P<0.05 or P<0.01) on the 3rd to 14th days after wounding. The content of the protein in the wound tissue in the taspine hydrochloride-treated group (2 mg•mL-1 ) was higher than that in the model group (P<0.05) on the 3rd to 7th days after wounding, and it arrived at the peak on the 7th day and gradually decreased to the normal level in skin tissue on the 14th to 21st days after wounding. The contents of hydroxyproline in the wound tissues in the taspine hydrochloride-treated groups were higher than that in the model group (P<0.05，P<0.01) on the 3rd to 21st days after wounding, and they arrived at the peak on the 14th day and at the normal level in skin tissues on the 21st day. Histopathology results showed that taspine hydrochloride could promote the formation of newly born blood capillaries in the early period of the wound healing.
3. The mechanism of taspine hydrochloride on enhancing wound healing.
3.1 Effect of taspine hydrochloride on the active of macrophage
The effect of taspine hydrochloride on the active of macrophage was assaied by phagocytosis. Over the concentration range 0.15～0.625 µg•mL-1, there was no obvious influence to macrophage. At the higher dose tested (1.25 μg•mL-1), there was a dose-dependent inhibition of the phagocytosis of neutral red.
3.2 Effect of taspine hydrochloride on fibroblast proliferation and autocrining growth factor by fibroblast cultured in vitro
To study the effect of taspine hydrochloride on fibroblast proliferation, fibroblasts were cultured in vitro. Over the concentration range 0.01~0.5 μg.mL-1 of taspine hydrochloride showed no effect on the change of lactate dehydrogenase activity and fibroblast proliferation.
Fibroblasts were cultured in vitro, and taspine hydrochloride was applied with different concentrations (0.05、0.1、0.2、0.4 and 0.5μg.mL-1) and time (24hr, 48hr, and 72hr) to influence their autocrine. The levels of transforming growth factor-β1and epidermal growth factor were determined by enzyme-linked immunoadsorbent assay (ELIAS). The results induced that: taspine hydrochloride could increase autocrine of TGF-β1 and EGF by fibroblasts, the action was reinforced with the increasing concentration of taspine hydrochloride and the extending of time.
3.3 Effect of taspine hydrochloride on cell growth factors and its receptor
Application of immunohistochemistry, in situ hybridization and image analysis to study the change of EGF、TGFβ1、TGF-β1R and TGFβ1mRNA expression in injured tissues. Evidence obtained in vitro: the expressions of EGF、TGFβ1、TGFβ1R and TGFβ1mRNA in the taspine hydrochloride treated-groups had a temporal character , they were observed from 3 to 21 days after injury and the highest expression was on the 7 day. Compared with bFGF group, the two had similar dynamic state change regularity. In the model group, their expressions were weaker.
3.4 Effect of taspine hydrochloride on the extracellular matrix in skin wounding healing
Application of immunohistochemistry and image analysis to study the effect of taspine hydrochloride on the expression of fibronectin, laminin,Ⅰ/Ⅲ type collagen in the graunlation tissue. The results induced that: taspine hydrochloride had discernible effects on the expression of fibronectin, laminin, andⅠtype collagen in injured tissues at days 3 to 21 after wounding. Taspine hydrochloride had no discernible effects on the expression of Ⅲ type collagen.
3.5 Effect of taspine hydrochloride on the MMP1, MMP9, TIMP1 in skin wounding healing
Application of immunohistochemistry, in situ hybridization and image analysis to study the effect of taspine hydrochloride on the expression of MMP1, TIMP1, TIMP1mRNA ,MMP9mRNA expression on wounding healing. The result inducted that: taspine hydrochloride had discernible effects on the expression of MMP1, TIMP1, TIMP1mRNA, MMP9mRNA in injured tissues at days 3 to 21 after wounding. The taspine hydrochloride-treated group showed an extensive distinguishable from the model group.
3.6 Effect of taspine hydrochloride in quantitative study of microvessels and iNOS in skin wounding
Application of immunohistochemistry in quantitative study of microvessels using the highly specific endothelial cell maker CD34 and factor Ⅷ.blood vessels were stained brownish yellow by immunohistochemistry. The number of microvessels in taspine hydrochloride -treated groups were higher at 3to 7days than that in model group（p<0.01，p<0.05）. The number of microvessels in taspine hydrochloride -treated groups were lower at days 14to 21 than that in model group(p<0.01，p<0.05). The expression of iNOS in the taspine hydrochloride-treated group was found to be higher than that of the model group (P<0.05) within 3 to 21days after the wounding, and there was a high iNOS amount compared with the model group at the days 7.
In summary, taspine hydrochloride can accelerate soft tissue repair in a dose-dependent manner at early phases of the process, the wound closure time was lesser, and improving wound contracting ability, accelerating the growth of newly born capillaries and raise the production of protein and collagen in wound tissue. It could accelerate the chemotaxis of fibroblast and inflammation cell, depositing of extracellular matrix and wound healing. The mechanism may be related to inhancing autocrining of EGF and TGF-β1, increasing or inhibiting the expression of cell growth factor at different phases in wound healing. Therefore taspine hydrochloride would have a good clinical value and it deserved to be further studied as a drug of promoting wound healing.