| Background:Scar is the inevitable product of the body wound healing. Pathological scar due to excessive scar hyperplasia, and causing abnormal shape and function. Hyperplastic Scar (HS) is one of the most common clinical pathological Scar, and causing appearance deformity and dysfunction of patients, which patients physical and psychological has brought great influence. Because of its specific mechanism is still not very clear, and the characteristics of easy relapse after treatment. Currently, there is no better methods of prevention and control to HS. Hyperplastic scar formation is more and more phase of a complex that including to coagulation, inflammation early, late inflammation, fibroblasts and collagen synthesis of migration, blood vessels, the epithelium, remodeling stage, etc. Traditional treatments include local pressurization, hormones, silicon gel type and concurrent chemo-radiation. Although its has obtained certain effect, but in the treatment of the HS at the same time, there will be side effects or adverse reactions, clinical application to limited. In traditional Chinese medicine theory, the HS thought to be due to blood indicates lag, meridian bizu, phlegmy wet stroke knot or combined, and based on promoting blood circulation to remove blood stasis, poison attack fights convergence, only strengthen the pain, acidity and disperse like knot ban method on hyperplastic scar prevention and control of a certain progress has been made. Though the report of a lot of methods of traditional Chinese medicine treatment of scar, but most of the existing medical way is unitary, limited dosage forms, more concentrated in a single drug or extracts research, and the curative effect is not exact, shortcomings and so on easy to relapse.Uygur medical theory is that body fluids is a basic material of human life and physiological activity process in all sorts of all sorts of nutrients produced in the liver total liquid is called humoral, divided into bile quality, quality of blood, phlegm and black bile. Them throughout the life, naturally in the body, have a great effect on health and disease. They constantly in the body use up, and constantly generate, maintain a certain balance. Four fluid out of the normal state of nature, on the quantity and quality change, become useless or damage to the human body fluids, called abnormal body fluids. Outside in the body under the action of various factors, human bladder fluid increase, function change, based on the body under the action of "hot" abnormal "combustion" eventually become the black bile, is common to many diseases uygur medicine pathological physiological features. Treatment mainly with abnormalities, black bile, mature agent (Abnormal Savda Munziq, ASMq) to adjust. ASMq part in prevention and treatment of tumor, diabetes, high blood pressure, etc, has achieved a certain effect. In vitro studies have shown that ASMq can inhibit tumor cell proliferation, promote cell apoptosis, and so on. Studies have shown that the skin proliferative scar is due to the excessive fibroblast proliferation and extracellular matrix caused by excessive deposition of collagen, also known as "skin fibroblasts benign tumor. This research in traditional medical theory under the guidance of the scientific hypothesis:abnormal hyperplastic scar skin is black bile quality caused by a local deposition of fluid in the skin, is the whole body black bile quality abnormal fluid local performance. Studied in this paper through the animal model of rabbit ear hyperplastic scar and human hyperplastic scar fibroblasts in vitro experiment, through the adoption of different concentration of ASMq intervention study, to test hypotheses, and further discuss the cellular and molecular mechanisms.Research shows that the hyperplastic scar is due to the excessive fibroblast proliferation and extracellular matrix caused by excessive deposition of collagen, the TGF-β is the important cytokine of hyperplastic scar formation, it is mainly through the TGF-β/Smad signaling pathways involved in regulating fibroblast proliferation, apoptosis, migration and collagen metabolism. TGF-P could trigger both can against Smad proteins mediated positive feedback regulation mechanism, which is rapid activate the Smad7promoter, induction of Smad7gene expression, through Smad7competitive occupy the TGF-β1, inhibit protein kinase receptor TGβR-phosphorylation and block the signal transduction of TGF-β, finally it can promote the extracellular matrix induced in wound deposition, and can promote the fibrosis, prompted the formation of hyperplastic scar.Because of hyperplastic scar is excessive fibroblast proliferation and extracellular mechanism caused by excessive deposition of collagen, accordingly inhibit fibroblasts proliferation, inhibit its collagen expression is the foundation of the prevention and treatment of hyperplastic scar. At the cellular level, by inhibiting cell activity to inhibit proliferation and promote apoptosis of the fibroblast can reduce the amount of scar fibroblasts in a certain extent, the prevention and treatment of hyperplastic scar; In cell ultrastructure changes in cells endoplasmic reticulum, ribosomes, closely associated with collagen expression of organelles such as mitochondria activity can be in a certain extent, have the effect that reduce scar collagen deposition; In the protein molecule level, because the TGF-β/Smad signaling pathway is one of the main channel of collagen, and TGF-β1is one of the important factors that promote cell proliferation, so by reducing the expression of TGF-β1, to increase the expression of Smad7can control the occurrence and development of hyperplastic scar. Objective:to experiment preliminary discussion of medicine ASMq hyperplastic scar and its mechanism of action in vitro, the influence of the drug for clinical used theory provide experimental and theoretical basis for the prevention and treatment of hyperplastic scar. Method:this research adopts the animal model of rabbit ear scar, hyperplastic scar fibroblasts in vitro culture, respectively from the level of histology, cytology and molecular biology, explored medicine ASMq of hyperplastic scar effect and its mechanism of action. The first part, the body experiment part, gastric gavage d medicine ASMq experimental study of the impact of rabbit ear hyperplastic scar.20New Zealand big-eared white rabbit by random number table method randomly divided into4groups, namely blank control group (normal saline) three experimental group respectively:(ASMq400mg/kg),800mg/kg (ASMq) and (ASMq1200mg/kg), each group5. In the wound after45days (preliminary experiments show that the scar hyperplasia peak), respectively, after materials to determine the indicators, slice prepared four experiments.(1) Through calculation of scar proliferation index (Hypertrophic Indes, HI), compared with the blank control group, so as to clear the experimental ASMq rabbit ear hyperplastic scar have any impact.(2) Through the group wound tissue section line within the scope of HE staining, the observation group rabbit ear scar arrangement of collagen and fibroblasts density, etc., further defined ASMq of rabbit ear hyperplastic scar hyperplasia of collagen and the influence of fibroblast proliferation.(3) Based on each line within the scope of the wound tissue Masson staining, observed between groups arranged collagen and collagen density, by using single factor analysis of variance analysis between multiple sets of collagen surface density, by comparing two q inspection, clear surface density of collagen have differences between groups.(4) Within the scope of rabbit ear wound tissue specimens were observed by transmission electron microscopy ultramicro structure (including collagen fibrils, mitochondria and endoplasmic reticulum cells structure), and analyses compared with the blank control group, further preliminary in ultrastructure level at different concentrations of medicine ASMq influence mechanism of rabbit ear hyperplastic scar. The second part, through the cultivation in vitro source of hyperplastic scar fibroblasts, four groups of three (a blank control group, experimental group):the blank control group, low concentration ASMq group (0.1mg/ml), concentration of ASMq group (0.4mg/ml), high concentration of ASMq group (0.7mg/ml). For each group collected samples for testing in the corresponding time points:(1) By the method of CCK-8, using enzyme standard meter testing ASMq fibroblast proliferation in vitro have impact.(2) The cell apoptosis analysis line apoptosis detection kit by flow cytometry analyzer, proposed explicitly different concentrations ASMq effects on fibroblast apoptosis.(3) Using the cell cycle detection kits, by flow cytometry analysis ASMq influence on fibroblast cell cycle, proposed explicitly ASMq blocking cell proliferation cycle of specific point in time.(4) Through the cell scratch migration experiment observe fibroblast migration ability, proposed explicitly ASMq different concentrations of fibroblast migration ability to have any effect.(5) By transmission electron microscope fibroblast synthesis ability of tropocollagen and endoplasmic reticulum, ribosomes, nucleus, mitochondria, cell structure, proposed further analysis clearly different concentration ASMq collagen and the expression of fibroblast apoptosis and proliferation mechanism. The third part, by adopting the method of western blot method based on analysis of TGF-β/Smad pathway ASMq source of hyperplastic scar fibroblasts molecular mechanism of cell proliferation and collagen expression effect. To clear different concentrations ASMq source of hyperplastic scar fibroblasts molecular mechanism of cell proliferation and collagen expression. Results:The first part, in vivo animal model of rabbit ear hyperplastic scar the experimental results show that the drug lavage ASMq can a certain extent, inhibit the occurrence development of hyperplastic scar, and in the certain concentration range has a concentration gradient dependence.(1) The general observations, as ASMq concentration gradually increases meanwhile the thickness gradually decreases, the hardness gradually softening, volume decreases, tend to be more calm, the color become weak of the hyperplastic scar.(2) The calculation result of scar proliferation index, compared with blank group (3.87±0.22), the experimental groups respectively (3.38±0.34),(2.63±0.15),(1.72±0.12) proliferative index (HI) reduce as ASMq concentration gradually increased (p<0.05).(3) by HE staining in concentration of1200mg/kg medicine ASMq of scar proliferation of fibroblasts after lavage observations, density (4022.5±189.3) compared with the blank group, the experimental group (3541.4±206.6) showed no obvious scar hyperplasia, the number of fibroblasts and density decrease (p<0.05).(4) By Masson staining in concentration of1200mg/kg medicine ASMq lavage after observation of scar hyperplasia of collagen fibers,(42.25±2.58) compared with the blank group, the experimental group (23.55±1.28) of hyperplastic scar collagen fiber surface density decreased significantly (p<0.05).(5) The different concentration of ASMq tem observation of hyperplastic scar fibroblasts ultrastructure observations; Compared with the blank group, along with the increasing concentration of experimental density of fibroblasts and collagen fiber surface density gradually reduce. The second part, the source of hyperplastic scar fibroblasts in vitro intervention experimental results show that in the certain concentration range ASMq can inhibit the role of fibroblast proliferation and promote apoptosis, which can inhibit cell ultrastructure analysis showed ASMq fibroblasts tropocollagen synthesis, inhibiting cell substructure (such as the endoplasmic reticulum, ribosomes, mitochondria, etc.), activity, promote cell apoptosis, and so on. The influence of the biological characteristics.(1) Original and extend the in vitro cell culture and as a result of cell frozen storage and recovery, the fibroblasts boundaries clear, the cell body is bigger, a prismatic or irregular triangle, the length of the two or three different protruding from the cytoplasm of protuberant, rich cytoplasm and cell membrane boundary clear, nucleolus with equal to double in size.(2) With the CCK-8method to detect the HSFs proliferation activity of the results, compared with the blank control group, different concentrations of ASMq group and positive control group5-Fu HSFs value-added activity significantly decreased, and the difference is statistically significant (p<0.05).(3)Using flow cytometry technology detection results between groups HSFs cell apoptosis, and the blank control group apoptosis rate of2.2%+0.59%, different concentration of ASMq apoptosis rate were14.1%±3.87%, while23.5%±3.22%,38.8%±3.14%, and43.7%±2.58%. Apoptosis rate ASMq group was obviously higher than that of control group, and the apoptosis rate showed a trend of increasing with ASMq concentration increase.(4) ASMq HSFs cell cycle impact as a result, compared with blank control group, different concentration of ASMq group HSFs in G2/M phase cells higher proportion in different degree, concentration dependence, that is between0.11.0mg/ml concentration, as ASMq concentration increases, G2/M phase cells was increased from13.1%to13.1%, the difference is statistically significant (p<0.05).(5) ASMq interference source of hyperplastic scar fibroblast migration results, compared with the blank group, experimental group respectively in Oh,24h,48h after the hyperplastic scar fibroblast migration ability over time and gradually reduced (p<0.05).(6) ASMq HSFs ultrastructure of impact as a result, from the macroscopic and observed at high magnification:0.4mg/ml group of fibroblast nuclei disappear,0.7mg/ml of fiber membrane a contraction,1mg/ml of fiber cells appeared apoptosis body relatively blank group.The third part, with different concentration of ASMq source of hyperplastic scar fibroblasts after intervention based on TGF-β/Smad pathway analysis, the results showed ASMq can inhibit the expression of TGF-β1, and promote the conservation of the expression of Smad7. In terms of molecular biological level, through detection method and western blot detection method.(1) ASMq of fibroblasts TGF-β1mRNA expression as a result, the influence of contrast with the blank group, ASMq with the increase of drug concentration, decrease of TGF-P mRNA expression (P<0.05). ASMq (2) The influence of the expression of fibroblast Smad7mRNA results, compared with the blank group, ASMq increased with the increase of drug concentration Smad7mRNA expression (P<0.05).(3) ASMq of fibroblasts TGF-β1protein expression as a result, the influence of contrast with the blank group, ASMq with the increase of drug concentration, decrease of TGF-β1protein expression (P<0.05).(4) ASMq effect on fibroblast Smad7protein expression as a result, compared with the blank group, AMSq increase with the increase of drug concentration Smad7protein expression (P<0.05). Conclusion:lavage ASMq can a certain extent to inhibit the occurrence development of rabbit ear hyperplastic scar, and dependency has concentration gradient in the certain concentration range; and the way to inhibit collagen synthesis and fibroblast proliferation. In a certain concentration range that can inhibit ASMq source of hyperplastic scar fibroblasts proliferation, promote cell apoptosis, and can inhibit cell ultrastructure. The result of analysis showed ASMq fibroblasts tropocollagen synthesis, inhibiting cell substructure (such as the endoplasmic reticulum, ribosomes, mitochondria, etc.), activity, promote cell apoptosis, and so on. ASMq can inhibit the one source of hyperplastic scar fibroblasts TGF-β1expression, to promote conservation of the expression of Smad7. In conclusion that ASMq can be based on TGF beta/Smad pathway inhibits fibroblast proliferation, promote cell apoptosis and inhibit the expression of collagen, the inhibiting the occurrence/development of hyperplastic scar, provides the hyperplastic scar prevention and control of new ideas. |
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