兰州大学机构库 >第二临床医学院
葛根素水凝胶对糖尿病大鼠创面愈合及血管形成的影响
Alternative TitleEffect of puerarin hydrogel on wound healing and angiogenesis in diabetic rats
周晶
Subtype硕士
Thesis Advisor张选奋
2023-05-30
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name医学硕士
Degree Discipline外科学
Keyword糖尿病创面 diabetic wound 葛根素 puerarin 创面愈合 wound healing 血管形成 vascular formation
Abstract

背景:糖尿病(DM)创面具有发病机制复杂、致残率高等特点,疗效较差、病程长、治疗费用高等给家庭和社会均造成巨大的负担。中药葛根提取物葛根素具有降低血糖、抗炎、抗氧化、修复神经和改善循环等功效,而且是迄今发现的第一种能够自然形成水凝胶的中药物质,它能够充分保留药理活性,有潜力成为促进创面愈合的新型辅料。

目的:探究葛根素水凝胶对DM大鼠皮肤创面愈合及血管形成的影响。

方法:1、DM大鼠模型的建立:45只SPF级SD大鼠,标准饲料喂养1周后持续予以高糖高脂饲料喂养,1%链脲佐菌素按40 mg/kg剂量腹腔注射,第7日时测尾静脉血糖,若血糖值≥16.7 mmol/L,并伴多饮、多食及多尿等症状视为DM大鼠造模成功。

2、葛根素水凝胶的配制:电子天平精确称取葛根素粉末10.0 mg置于1.0 mL PBS中,酒精灯加热至100 ℃,直至葛根素粉末完全溶解,室温下冷却5-10 min即可形成1%葛根素水凝胶。2%和4%葛根素水凝胶同法制备。

3、DM创面模型的建立及分组:36只DM大鼠,每只腹腔注射6.0 mL/kg的1%戊巴比妥钠溶液麻醉,电推仔细剔除背部毛发,设计直径2.0 cm的圆形创面,按标记切开至肌层,用组织剪剪除全层皮肤。随机数表法分为对照组、1%葛根素组、2%葛根素组和4%葛根素组。对照组创面以PBS湿敷处理,葛根素组创面以无菌棉签涂抹不同浓度葛根素水凝胶,涂抹厚度约2.0 mm,每天1次,连续14天,涂抹范围至少超出创缘2.0 mm,用无菌纱布覆盖后妥善固定。

4、创面愈合情况观察和创面愈合率计算:观察创面愈合过程有无明显出血、污染或感染等。第3、7和14天拍照记录。使用Image pro plus 6.0软件测量创面面积,取同一时刻点各组间大鼠创面面积求平均值。按“创面愈合率=(初始创面面积-实际创面面积)/初始创面面积×100%”公式计算创面愈合率。

5、组织学检测:第3、7和14天,每组取3只,紧贴大鼠背部创面边缘切开至肌层,完整切取创面组织,根据需要将组织块分别保存于4%多聚甲醛溶液中或-80 ℃冰箱中。氧化酶法测定创面组织葡萄糖含量,HE及Masson染色观察创面组织学变化,免疫组织化学法做血管内皮细胞生长因子(VEGF)和CD31染色,显微镜下拍照后利用Image pro plus 6.0软件计算积分光密度(IOD)及微血管密度(MVD)。Western Blot半定量法检测各组创面组织中VEGF蛋白的表达。

6、统计学处理和图像分析:使用SPSS 26.0软件进行数据统计处理,Image pro plus 6.0软件用于图像分析和计算。体重、血糖、组织葡萄糖含量、创面愈合率、胶原纤维面积、IOD及MVD等计量资料用`x±s 表示,多组间比较用F检验,各组间比较采用LSD法。以双侧P<0.05视为存在统计学意义。

结果:1、体重及血糖:各组大鼠初始体重及血糖无统计学差异(P>0.05)。各葛根素组大鼠在DM创面造模后体重变化趋势与对照组无统计学差异,均表现为第1周出现体重增速减缓,第2周体重缓慢下降(P>0.05),各葛根素组血糖值与对照组无统计学差异(P>0.05),均表现为在两周内均出现小幅波动现象(波动范围不超过3.0 mmol/L)。4%葛根素组各大鼠在DM创面造模后体重变化和血糖水平与其余低浓度葛根素组无统计学差异(P>0.05)。

2、创面愈合情况:各组创面均随时间逐渐缩小。与对照组比较,各葛根素组创面较早形成新鲜肉芽组织,愈合明显加快(P<0.05);与其他低浓度葛根素组比较,各时刻点均以4%葛根素组愈合率最高(P<0.05)。

3、创面组织葡萄糖含量测定:各葛根素水凝胶处理前后创面组织葡萄糖含量与对照组无统计学差异(P>0.05);创面组织葡萄糖含量在各浓度葛根素组间也未见变化(P>0.05)。

4、组织学变化:HE及Masson染色结果显示,第3天时,对照组无血管及胶原纤维形成,各葛根素组有初期毛细血管形成,胶原纤维较稀疏,其中以4%葛根素组毛细血管形成较明显,但管壁结构欠成熟。第7天时,对照组血管形成仍较困难,胶原纤维较稀疏;各葛根素组有不同程度的薄壁毛细血管形成及胶原纤维分布,其中1%和2%葛根素组呈现新生样毛细血管,但管壁结构尚欠规则,4%葛根素组已呈现早期的血管改建趋势,管壁增厚且趋于规则,胶原纤维较丰富。第14天时,对照组形成少量薄壁毛细血管,管腔不规则,胶原较前稍致密;各葛根素组毛细血管数量增多,并出现管腔狭窄、管壁增厚及胶原纤维丰富等愈合后期改变,以4%葛根素组愈合趋势最明显。

5、VEGF和CD31免疫组织化学染色:随时间增加,各组创面VEGF的IOD和MVD均逐渐增加,各葛根素组VEGF的IOD和MVD增幅较大(P<0.05)。与其他低浓度葛根素组比较,同一时刻点时4%葛根素组VEGF的IOD和MVD均最高(P<0.05)。

6、Western blot半定量检测VEGF蛋白表达:第3、7天时,1%葛根素组VEGF蛋白表达与对照组无统计学差异(P>0.05),2%和4%葛根素组间VEGF蛋白表达高于对照组(P<0.05);4%葛根素组VEGF蛋白表达高于其他低浓度葛根素组(P<0.05)。第14天时,各葛根素组VEGF蛋白表达均高于对照组(P<0.05);4%葛根素组的VEGF蛋白表达高于其他低浓度葛根素组(P<0.05)。

结论:葛根素水凝胶呈浓度依赖性促进DM创面组织表达VEGF和CD31,诱导微血管形成,促进创面组织胶原纤维的沉积,从而促进DM创面愈合。

Other Abstract

Background: The wound of diabetes mellitus (DM) has the characteristics of complex pathogenesis and high disability rate. The poor curative effect, the long course of the disease, the high cost of treatment and other factors have caused a huge burden on the family and society. Puerarin, a Chinese medicine extract of pueraria root, has the effects of lowering blood sugar, anti-inflammatory, antioxidant, repairing nerves and improving circulation. It is also the first Chinese medicine substance that can form hydrogels naturally. It can fully retain pharmacological activity and has the potential to be a new dressing to promote wound healing.

Objective: To explore the effect of puerarin hydrogel on skin wound healing and vascular formation in DM rats.

Methods: 1. Establishment of DM rat model: 45 SPF SD rats were fed with 1 week of standard feed and continuously fed high-glucolipid diet. 1% streptozotocin was injected intraperitoneally and tail vein blood glucose was measured at day 7. If blood glucose value was 16.7 mmol/L, polydipsia, polyphagia and polyuria were considered as successful in DM rats.

2.Preparation of puerarin hydrogel: Electronic scale accurately weighed puerarin powder 10.0 mg and placed it in 1 mL PBS. Heat the alcohol lamp to 100℃ until puerarin powder is completely dissolved. 1% puerarin hydrogel can be formed by cooling at room temperature for 5-10 min. 2% and 4% puerarin hydrogels were prepared by the same method.

3.Establishment and grouping of DM wound model: 36 DM rats were anesthetized by intraperitoneal injection of 6.0 mL/kg 1% sodium pentobarbital solution. Use electric shear to carefully remove back hair. A circular wound with a diameter of 2.0 cm was designed and cut to the muscle layer according to the marks. The whole skin layer was removed with tissue scissors. They were divided into control group, 1% puerarin group, 2% puerarin group and 4% puerarin group according to random number table method. The wound of control group was treated with PBS wet compress, while puerarin group was treated with puerarin hydrogel of different concentrations applied with sterile cotton swabs. The coating thickness should be about 2.0mm once a day for 14 consecutive days, and the coating scope should be at least 2.0mm beyond the wound edge. The coating should be covered with sterile gauze and properly fixed.

4.Observation of wound healing and calculation of wound healing rate: Observe whether there is obvious bleeding, contamination or infection during wound healing. The photos were taken on days 3, 7 and 14. Image pro plus 6.0 software was used to measure the wound area. The wound area of each group was averaged at the same time point. The wound healing rate was calculated according to the formula "wound healing rate = (initial wound area - actual wound area)/initial wound area ×100%".

5.Histological examination: On the 3rd, 7th and 14th day, 3 rats from each group were cut close to the wound edge of the back of the rats to the muscular layer, and the wound tissue was completely cut. Store tissue blocks in 4% paraformaldehyde solution or -80℃ refrigerator as needed. Glucose content in wound tissue was measured by oxidase method. Histological changes of the wound were observed by HE and Masson staining. Vascular endothelial cell growth factor (VEGF) and CD31 were stained by immunohistochemistry. Integrated optical density (IOD) and microvascular density (MVD) were calculated using Image pro plus 6.0 software after taking pictures under the microscope. Western Blot Semi-quantitative was used to detect the expression of VEGF protein in wound tissues of each group.

6.Statistical processing and image analysis: SPSS 26.0 software was used for data statistical processing. Image pro plus 6.0 software is used for image analysis and calculation. Measurement data such as body weight, blood glucose, tissue glucose content, wound healing rate, collagen fiber area, IOD and MVD were expressed by `x±s . F test was used for comparison between groups. LSD method was used for comparison between groups. Bilateral P < 0.05 was considered statistically significant.

Results: 1. Weight and blood sugar:There was no statistical difference in initial body weight and blood glucose in each group (P>0.05). The trend of body weight after the DM wound was not different from the control group, the weight growth slowed down in the first week, and the weight decreased slowly in the second week (P>0.05). The blood glucose values of each puerarin group were not statistically different from the control group (P>0.05), and all showed small fluctuations within two weeks (fluctuation range was no more than 3.0 mmol/L). Body weight changes and blood glucose levels in the 4% puerarin group were not significantly different from the remaining low-concentration puerarin group (P>0.05).

2.Wound healing: The wound surface of each group decreased gradually with time. Compared with the control group, puerarin groups formed fresh granulation tissue earlier and healed significantly faster (P<0.05). Compared with other low concentration puerarin groups, 4% puerarin group had the highest healing rate at all time points (P<0.05).

3.Measurement of glucose content in wound tissue: There was no statistical difference in glucose content between wound tissue before and after each puerarin hydrogel treatment and the control group (P>0.05). The glucose content of wound tissue was not changed among puerarin groups (P>0.05).

4.Histological change: The results of HE and Masson staining showed that no blood vessels or collagen fibers were formed in the control group on the 3rd day, while the puerarin groups had initial capillary formation and collagen fibers were sparse. The capillary formation was more obvious in 4% puerarin group, but the structure of tube wall was immature. On the 7th day, blood vessel formation was still difficult in the control group, and collagen fibers were sparse. There were different degrees of thin-wall capillary formation and collagen fiber distribution in puerarin groups, among which 1% and 2% puerarin groups showed new capillaries, but the wall structure was not regular. 4% puerarin group showed an early trend of vascular remodeling, the tube wall thickened and tended to be regular, and collagen fibers were abundant. On the 14th day, a small number of thin-walled capillaries were formed in the control group, with irregular lumen and slightly denser collagen. The number of capillaries in puerarin groups increased, and there were some late healing changes such as lumen stenosis, tube wall thickening and collagen fiber richness. The healing trend of 4% puerarin group was the most obvious.

5.Immunohistochemical staining of VEGF and CD31: Over time, the IOD and MVD of VEGF increased gradually in each group. The IOD and MVD of VEGF increased significantly in puerarin groups (P<0.05). Compared with other low concentration puerarin groups, the IOD and MVD of VEGF in 4% puerarin group were the highest at the same time point (P<0.05).

6.Western blot semi-quantitative detection of VEGF protein expression: On days 3 and 7, VEGF protein expression in the 1% puerarin group was not significantly different from the control group (P>0.05). The protein expression of VEGF in 2% and 4% puerarin groups was higher than that in control group (P<0.05). The protein expression of VEGF in 4% puerarin group was higher than that in other low concentration puerarin groups (P<0.05). On day 14, the protein expression of VEGF in puerarin groups was higher than that in control group (P<0.05). VEGF protein expression in 4% puerarin group was higher than that in other low concentration puerarin groups (P<0.05).

Conclusion: Puerarine hydrogel promoted the expression of VEGF and CD31 in DM wound tissue in a concentration-dependent manner, induced the formation of microvessels, promoted the deposition of collagen fibers in the wound tissue, and thus promoted the healing of DM wound.

MOST Discipline Catalogue医学 - 临床医学 - 外科学
URL查看原文
Language中文
Other Code262010_220200904920
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/536409
Collection第二临床医学院
Affiliation
兰州大学第二临床医学院
Recommended Citation
GB/T 7714
周晶. 葛根素水凝胶对糖尿病大鼠创面愈合及血管形成的影响[D]. 兰州. 兰州大学,2023.
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