臭氧水对感染性创面抗炎修复作用的初步实验研究
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摘要
研究背景
臭氧(Ozone, O3)具有强氧化性与不稳定性,在常温常压空气中易还原为氧气(半衰期45分钟)。臭氧可溶于水,在常温常压下臭氧在水中的溶解度比氧高约13倍,比空气高25倍。但臭氧在水中的分解速度比空气中快的多。如水中臭氧浓度为6.25×10-5 mol/L(3mg/l)时,其半衰期为5-30min,但在纯水中分解速度较慢,如在蒸馏水或自来水中的半衰期大约是20min(20℃),然而在二次蒸馏水中,经过85min后臭氧分解只有10%,若水温接近0℃时,臭氧会变得更加稳定。
医用臭氧(Medical ozone, O2-O3)是利用医用纯氧制备的臭氧与纯氧的混合气体,具有较强的氧化作用,能通过氧化还原作用迅速杀灭细菌,为广谱有效的强杀菌剂。随着对医用臭氧的生物药学特性认识的深入研究,臭氧在医疗界中被广泛应用,在欧洲等地医用臭氧广泛用于治疗糖尿病足、创伤及皮肤溃疡的愈合、脑中风、溃疡性结肠炎、关节炎、椎间盘突出症等疾病,其临床疗效得到了充分肯定。臭氧溶于水后形成的臭氧水具有更强的消毒杀菌功能和分解催化作用,能把有机物、重金属分解或转化为对人体无害的物质。针对臭氧水这一特性,国内外学者在临床中大量应用臭氧消毒水,对感染性创面清创,口腔含漱,阴道冲洗消毒等方面取得了良好的效果,而且臭氧水制备设备体积小,制备简单迅速,在冰箱中保存可使用3-4天,相对于传统的消毒剂有明显的优势。
临床应用结果表明臭氧治疗具有显著的优越性,但为何迟迟未能真正推广呢?其最根本的原因就是其基础研究远远落后于临床实践,许多作用及机制尚未明了,限制了其临床治疗领域的扩展；尤其是目前尚缺乏有关臭氧治疗安全性以及使用浓度的系统的基础研究,成为其发展的瓶颈。因此,医用臭氧安全性及应用浓度范围的相关研究研究显得尤为重要。
目前关于臭氧疗法安全性的研究集中在臭氧对血液及血细胞特别是红细胞的影响方面。臭氧有强烈的氧化作用,接触一定浓度、一定的持续时间会造成机体的损害。虽然血浆中有缓冲系统以及一系列的抗氧化剂存在(如维生素C、尿酸、巯基蛋白、糖蛋白等),造成臭氧的作用被中和、活性迅速降低,但仍有部分的臭氧在血浆中会生成活性氧(ROS),在体外血液实验中,ROS可以触发若干生物化学通路,生成的ROS在0.5-1.0分钟内被血浆中的抗氧化系统中和；在接触并溶解在血浆中时,臭氧迅速和抗氧化剂及多不饱和脂肪酸发生反应,级联生成一系列复合物,如过氧化氢、脂质过氧化产物等(其中作用较明显的4—羟壬烯醛能够激活血液、内皮细胞和实质细胞的某些功能,从而发挥对各种疾病的生理功能调节及治疗的效应)。Bocci等的研究表明,医用臭氧浓度小于80μg／ml,血液及红细胞中的缓冲系统、抗氧化系统可以抵抗臭氧对组织、细胞的损伤作用,从而发挥臭氧的治疗作用；当臭氧浓度大于80μtg／ml,则易引起组织细胞的损伤,随着臭氧浓度的增加,损伤作用更加明显。
但是以往针对臭氧的研究主要集中在臭氧气体的抗炎、镇痛、免疫调节,臭氧水的研究较少,而且主要集中在临床效果这一方面。因此,有必要更进一步探讨,与臭氧气体相比较,臭氧水的浓度不同,其作用效果是否不同?不同浓度的臭氧水作用于机体,机体的局部组织发生了何种变化,是何种机制导致了这种变化?这些问题是临床臭氧治疗医师共同关注的问题,在一定程度上影响患者对臭氧治疗的接受程度,成为医用臭氧治疗推广应用的一个阻碍。但目前国内外尚未见相关报道。
研究目的
1.不同浓度臭氧水对感染性创面的抗炎与修复作用的效果；
2.臭氧水治疗感染性创面后,对内源性生长因子含量的影响,探讨臭氧水抗炎修复作用的机制。
研究方法
1.臭氧水对感染性创面抗炎修复作用部分
将50只vista大鼠随机分成5组,洗必泰治疗组、低、中、高浓度臭氧水治疗组、对照组(不做特殊处理)各10只。首先在大鼠背部造成实验性感染性伤口,感染成功后48 h进行治疗,每天1次,9天为1疗程。在治疗前、治疗后3、6、9 d后分别对感染伤口的变化进行观察,抽取鼠尾静脉血检查白细胞计数,切取皮肤、肌肉组织标本进行光镜病理分析。
2.臭氧水对感染性创面修复作用与内源性细胞因子含量变化的关系部分
在第一部分的实验基础上,同时在治疗前、治疗后3、6、9 d后,分别切取创面的皮肤、肌肉组织标本,用elisa法分别检测其细胞因子(TNF-α、EGF)的含量,并探讨这两种细胞因子含量变化与臭氧水对创面修复作用的关系
3.统计学分析：所得数据用SPSS13.0软件行统计学处理,对各组的白细胞总数、EGF含量、TNF-α含量,分别在感染前、后以及治疗后各阶段比较采用General Linear Model中的repeated measures进行分析,检验水准为a=0.05,-0.05表示差异有统计学意义或有显著性差异；在治疗各个阶段,治疗组与对照组比较以及洗必泰组与臭氧水治疗组比较,以及治疗3、6、9d后,不同浓度臭氧水治疗组间两两比较,采用Compare Means中的one-wayanova进行分析(文件需要Splitfile),检验水准为a=0.05,P≤0.05表示差异有统计学意义或有显著性差异。
研究结果
1.人工感染伤口所见：治疗后比较,臭氧水治疗组疗效远优于对照组以及洗必泰组,其中10mg／L与20mg／L臭氧水组疗效又优于5mg／L组。
2.白细胞总数变化：治疗3、6、9d后,10mg／L与20mg／L组白细胞总数无显著性差异(P>0.05),而前两者与5mg／L组在治疗后3、6d后有显著性差异(P<0.05)。治疗后9d,三种浓度的臭氧水治疗组白细胞的总数差异不显著。
3.光镜所见：治疗3d后,臭氧水治疗组炎症病理减轻,可见新生表皮细胞,以10mg／L组与20mg／L组明显；对照组炎症改变加重,出现肌纤维坏死。治疗6d后,洗必泰组炎症减轻不明显；臭氧水治疗组炎症病理进一步减轻,其中10mg／L组与20mg／L组上皮结构修复完整；对照组皮肤、肌肉坏死表现进一步加重。治疗后9d,洗必泰组炎症表现减轻,开始出现部分表皮细胞连接；臭氧水治疗组只有轻微的炎症表现,无渗出,10mg／L组与20mg／L组大部分恢复正常皮肤,肌纤维肉芽组织大量生长；对照组出现慢性炎症的表现。
4.EGF含量变化：在治疗各个阶段,治疗组与对照组比较以及洗必泰组与臭氧水治疗组比较均有显著性的差异(P<0.05),治疗3、6、9d后,10mg／L与20mg／L组无显著性差异(P>0.05),而前两者在治疗后3、6d后,EGF含量高于5mg／L组。而在治疗后9d,三种浓度的臭氧水治疗组EGF含量差异不显著。
5.TNF-α含量变化：在治疗各个阶段,治疗组与对照组比较以及洗必泰组与臭氧水治疗组比较均有显著性的差异,治疗3、6、9d后,10mg／L与20mg／L组无显著性差异,而前两者与5mg／L组在治疗后3、6d后有显著性差异,在治疗后第9d差异不显著,5mg／L组TNF-α含量高于前两者。
结论
1.中、高浓度臭氧水的抗炎消毒效果优于低浓度组,而中与高浓度组的效果没有明显差异,提示在临床应用中,10-20mg／L的臭氧水已足够控制甚至消灭创面感染。
2.EGF与大鼠创面的抗炎修复结局成正相关性,臭氧水治疗能促进创面组织中的EGF含量的升高,而且与其浓度有关,其中以10mg／L与20mg／L浓度组最佳,EGF含量在治疗过程中逐步增加。
3.臭氧水治疗对创面中TNF-α含量影响异于EGF,臭氧水治疗组中TNF-α含量普遍低于洗必泰组以及对照组,其中10 mg／L与20 mg／L浓度臭氧水治疗组中的TNF-α含量最低。臭氧水在治疗感染性创面的过程中降低了TNF-α含量,从而抑制了NF-KB系统的活性,达到抗炎修复组织的目的。
Background
Ozone (O3) with strong oxidation and instability, in the air at normal temperature and pressure easily reduce to oxygen (has half-life about 45 minutes). Ozone can soluble in water, in the air at normal temperature and pressure,it's solubility in water is 13 times higher than oxygen,and 25 times higher than air.However,the speed of decomposition in water is much faster than that in the air.For example,if the concen-tration of ozone in water is 6.25×10-5 mol/L(3mg/l), the half-decay period is 5～30min,but it decompose slowly in purified water, the half-decay period is 20min in distilled water or tap water.When in the redistilled water,only 10%of ozone decompose after 85 min,if the temperature of the water close to 0℃,ozone will be more stability.
Medical ozone (O2-O3) is the mixture of ozone and pure oxygen,which can quickly kill bacterium under oxidation-reduction reaction,it's consider as one kind of broad and utility bactericide duo to its stronge oxygenation ability. Along with recognizant and lucubrate oxygen-ozone to characteristic of biopharmacy, oxygen-ozone gradually expand to midecal area. In Europe, medical ozone are widely used for the treatment of diabetic foot wide, trauma and the healing of skin ulcers, stroke, ulcerative colitis, arthritis, prolapse of intervertebral disc, et al, its clinical efficacy has been fully affirmed.Ozone water, solubled in water by ozone gas,is more stronger in sterilizing, decomposition and promotion, organics and heavy metalcan be break dowm or transform into harmless material.Considering the character of ozone water, it is widely used in debrideing infected wound,mouthwash, vagina washing by hakeem domestic and overseas,and gain wonderful effect.On the other side,the facilities of prepare ozone water are small, the process is simple and quick,it couble bo conservation in the frige for 3-4days,all of which make ozone water much superior than traditional antiseptics.
Clinical results show that ozone therapy has significant advantages, but why it has not really promoted, and even be refused by many doctors and patients? The most fundamental reason is that the basic research of ozone therapy is lagging far behind in clinical practice, many roles and mechanisms of the therapy still unknown, what limit its expansion of the field of clinical treatment. In particular, the lack of basic research on safety of the ozone therapy blocks its development. So the safety of medical ozone therapy studies appears to be every important.
At present, the related research focuses on the ozone in the blood cells, in particular the impact of erythrocytes. Ozone has a strong sense of oxidation, contact a certain concentration and duration of ozone will cause the body damage. Most research on the ozone focus on the affection on blood erythrocytes.
Although plasma has series of buffer system, such as antioxidant enzymes and glutathione, to prevent accumulation of the superoxide anion and hydrogen peroxide, which may produce alterations of tissue structure secondary to cell membrane degradation, but there is still part of the ozone in the plasma generates reactive oxygen species (ROS). In vitro experiments, the ROS can trigger a number of biochemical pathways. ROS degradation in 0.5 to 1.0 minutes by antioxidant system in the plasma. As soon as contact and dissolved in the plasma, ozone react with anti and polyunsaturated fatty acids, and cascade generated a series of complexes which had been definited, such as hydrogen peroxide and lipid peroxidation products. Studies by Bocci, etc, suggested that when concentration of medical ozone was less than 80μg/ml, buffer systems and anti-oxidation system in the blood and red blood cell, can resistant the ozone oxidization and avoid cells and tissue damage, thus ozone can play a role as drug. When ozone concentrations more than 8μg/ml, it causes tissue damage easily, with the concentration of ozone increasing, there is more obvious damage.
However, the past reseashs are mainly concentrate on the anti-inflammatory, analgesia and immunoloregulation,the fewer studies on ozone water are localize on clinic.For this reason,it's necessary to find out,compare to ozone water,does different density of ozone water has different effert? What is the transmutation of organism contribute to ozone water,and what is the mechanicsm? Clinical physicians use ozone for treatments common concern these questions and the latter affect patients on the acceptability of the ozone treatment in a certain extent, and block the development of ozone therapy. But we have not yet seen relevant reports.
Purpose
1.To study the healing effect of different density of ozone water on the infected wound;
2.To explore the changes in endogenous growth factors and their relation with wound healing by ozone water.
Method
1. First part:The role of ozone water on antiinflammatory and reparation of infected wound.
50 vista rats were randomly divided into five groups:chlorhexidine treatment group、5mg/L、10mg/L、20mg/L ozone water treatment group and contrast group(without any special treatment). The back of rats were artificially formed the infected wound. After 48 hours of infection, the chlorhexidine or ozone water was given, once a day,for 9 times. Before and after the 3rd,6th,9th day of treatment, the alteration of infected wound was observed. The WBC of tail venous blood was detected and the incised skin and musle tissue was lstopathologically analysed by light microscope.
2. Second part:The changes in endogenous growth factors and their relation with wound healing by ozone water
Based on the first part of the experiment, before and after the 3rd,6th,9th day of treatment, harvested the muscle and skin tissue samples of the wound.The amounts of endogenous epidermal growth factor(EGF) and tumor necrosis factor-α(TNF-α) in infected wound were determined and their relation with the wound healing by ozone water were studied.
3. Statistics analysis:The experimental data was analyzed by the software of SPSS 13.0, and an associated probability of 5%(P<0.05) was considered significant.
Results
1. Artificial infected woud findings:Compare with post-treatment, ozone water have the better effect to promote the healing of infected wound, especially the result of 10mg/L and 20mg/L are better.
2. Total white blood cell changes:After the 3rd,6th,9th day of treatment, no significant difference in the total number of white blood cell betweem 10mg/L and 20mg/L ozone water treatment group (P> 0.05).But the first two have significant difference with 5mg/L ozone water treatment group after the 3rd,6th day of treatment.After the 9th day of treatment, the total number of white blood cells was no significant difference amon three kinds of concentrations of ozone water treatment group.
3. Light microscopy findings:After the 3th day of treatment, Ozone water treatment group reduce inflammation in pathological, newborn epidermal cells can be seen, especially in the result of 10mg/L and 20mg/L groud; Inflammatory changes in the contrast group was increased, muscle fiber necrosis can be seen. After the 3th day of treatment,reduce inflammation in pathological of chlorhexidine treatment groud was not obvious, the inflammatory pathology of ozone water treatment group were further reduced. Epithelial repair integritily in the 10mg/L and 20mg/L groud; Skin, muscle necrosis increased in the contrast group. After the 9th day of treatment, inflammation in pathological of chlorhexidine treatment groud was reduced, Some epidermal cells appeard to connect; Ozone water treatment group have only mild inflammation and no effusion, 10mg/L and 20mg/L groud mostly return to normal skin, The growth of granulation tissue and muscle fibers were significant, The contrast group appeared to chronic inflammation.
4.Content changes of EGF:In all stages of treatment, the treatment group compared with the control group and the chlorhexidine group compared with the ozone water treatment group,there were significantly difference (P<0.05). After the 3rd,6th and the 9th day of treatment,ther was no significant difference betweem 10mg/L and 20mg/L groud (P> 0.05).But the amounts of EGF in the first two were much greater than those in 5mg/L groud after the 3rd,6th day of treatment. After the 9th day of treatment, the Content changes of EGF were no significant difference amon three kinds of concentrations of ozone water treatment group.
5. Content changes of TNF-α:In all stages of treatment, the treatment group compared with the control group and the chlorhexidine group compared with the ozone water treatment group,there were significantly difference (P<0.05). After the 3rd,6th and the 9th day of treatment,ther was no significant difference betweem 10mg/L and 20mg/L groud (P> 0.05).But the amounts of EGF in the first two were significant different from 5mg/L groud,5mg/L groud was higher.
Conclusion
1. Medium and high concentration of ozone water groud have the better effect to promote the healing of infected wound than the low one,and the first have similar effect,which indicated that 10-20mg/L ozone water is Enough to control and even eradication of wound infection.
2. There is a positive correlation betweem EGF and wound repairment in rats,ozone water treatment can increase the content of EGF,and have a relationship with its concentration,10mg/L and 20mg/L groud have the best effect,the content of EGF gradually increased during the treatment process.
3. Opposite to the EGF,the content of TNF-αin ozone water treatment groud was lower than that in the chlorhexidine treatment group and contrast group,10mg/L and 20mg/L groud were the lowest. Ozone water treatment can reduce the content of TNF-α, thereby inhibiting the activity of the NF-κB system,finaly reach the purpose of antiinflammatory and reparation.
臭氧(Ozone, O3)具有强氧化性与不稳定性,在常温常压空气中易还原为氧气(半衰期45分钟)。臭氧可溶于水,在常温常压下臭氧在水中的溶解度比氧高约13倍,比空气高25倍。但臭氧在水中的分解速度比空气中快的多。如水中臭氧浓度为6.25×10-5 mol/L(3mg/l)时,其半衰期为5-30min,但在纯水中分解速度较慢,如在蒸馏水或自来水中的半衰期大约是20min(20℃),然而在二次蒸馏水中,经过85min后臭氧分解只有10%,若水温接近0℃时,臭氧会变得更加稳定。
医用臭氧(Medical ozone, O2-O3)是利用医用纯氧制备的臭氧与纯氧的混合气体,具有较强的氧化作用,能通过氧化还原作用迅速杀灭细菌,为广谱有效的强杀菌剂。随着对医用臭氧的生物药学特性认识的深入研究,臭氧在医疗界中被广泛应用,在欧洲等地医用臭氧广泛用于治疗糖尿病足、创伤及皮肤溃疡的愈合、脑中风、溃疡性结肠炎、关节炎、椎间盘突出症等疾病,其临床疗效得到了充分肯定。臭氧溶于水后形成的臭氧水具有更强的消毒杀菌功能和分解催化作用,能把有机物、重金属分解或转化为对人体无害的物质。针对臭氧水这一特性,国内外学者在临床中大量应用臭氧消毒水,对感染性创面清创,口腔含漱,阴道冲洗消毒等方面取得了良好的效果,而且臭氧水制备设备体积小,制备简单迅速,在冰箱中保存可使用3-4天,相对于传统的消毒剂有明显的优势。
临床应用结果表明臭氧治疗具有显著的优越性,但为何迟迟未能真正推广呢?其最根本的原因就是其基础研究远远落后于临床实践,许多作用及机制尚未明了,限制了其临床治疗领域的扩展；尤其是目前尚缺乏有关臭氧治疗安全性以及使用浓度的系统的基础研究,成为其发展的瓶颈。因此,医用臭氧安全性及应用浓度范围的相关研究研究显得尤为重要。
目前关于臭氧疗法安全性的研究集中在臭氧对血液及血细胞特别是红细胞的影响方面。臭氧有强烈的氧化作用,接触一定浓度、一定的持续时间会造成机体的损害。虽然血浆中有缓冲系统以及一系列的抗氧化剂存在(如维生素C、尿酸、巯基蛋白、糖蛋白等),造成臭氧的作用被中和、活性迅速降低,但仍有部分的臭氧在血浆中会生成活性氧(ROS),在体外血液实验中,ROS可以触发若干生物化学通路,生成的ROS在0.5-1.0分钟内被血浆中的抗氧化系统中和；在接触并溶解在血浆中时,臭氧迅速和抗氧化剂及多不饱和脂肪酸发生反应,级联生成一系列复合物,如过氧化氢、脂质过氧化产物等(其中作用较明显的4—羟壬烯醛能够激活血液、内皮细胞和实质细胞的某些功能,从而发挥对各种疾病的生理功能调节及治疗的效应)。Bocci等的研究表明,医用臭氧浓度小于80μg／ml,血液及红细胞中的缓冲系统、抗氧化系统可以抵抗臭氧对组织、细胞的损伤作用,从而发挥臭氧的治疗作用；当臭氧浓度大于80μtg／ml,则易引起组织细胞的损伤,随着臭氧浓度的增加,损伤作用更加明显。
但是以往针对臭氧的研究主要集中在臭氧气体的抗炎、镇痛、免疫调节,臭氧水的研究较少,而且主要集中在临床效果这一方面。因此,有必要更进一步探讨,与臭氧气体相比较,臭氧水的浓度不同,其作用效果是否不同?不同浓度的臭氧水作用于机体,机体的局部组织发生了何种变化,是何种机制导致了这种变化?这些问题是临床臭氧治疗医师共同关注的问题,在一定程度上影响患者对臭氧治疗的接受程度,成为医用臭氧治疗推广应用的一个阻碍。但目前国内外尚未见相关报道。
研究目的
1.不同浓度臭氧水对感染性创面的抗炎与修复作用的效果；
2.臭氧水治疗感染性创面后,对内源性生长因子含量的影响,探讨臭氧水抗炎修复作用的机制。
研究方法
1.臭氧水对感染性创面抗炎修复作用部分
将50只vista大鼠随机分成5组,洗必泰治疗组、低、中、高浓度臭氧水治疗组、对照组(不做特殊处理)各10只。首先在大鼠背部造成实验性感染性伤口,感染成功后48 h进行治疗,每天1次,9天为1疗程。在治疗前、治疗后3、6、9 d后分别对感染伤口的变化进行观察,抽取鼠尾静脉血检查白细胞计数,切取皮肤、肌肉组织标本进行光镜病理分析。
2.臭氧水对感染性创面修复作用与内源性细胞因子含量变化的关系部分
在第一部分的实验基础上,同时在治疗前、治疗后3、6、9 d后,分别切取创面的皮肤、肌肉组织标本,用elisa法分别检测其细胞因子(TNF-α、EGF)的含量,并探讨这两种细胞因子含量变化与臭氧水对创面修复作用的关系
3.统计学分析：所得数据用SPSS13.0软件行统计学处理,对各组的白细胞总数、EGF含量、TNF-α含量,分别在感染前、后以及治疗后各阶段比较采用General Linear Model中的repeated measures进行分析,检验水准为a=0.05,-0.05表示差异有统计学意义或有显著性差异；在治疗各个阶段,治疗组与对照组比较以及洗必泰组与臭氧水治疗组比较,以及治疗3、6、9d后,不同浓度臭氧水治疗组间两两比较,采用Compare Means中的one-wayanova进行分析(文件需要Splitfile),检验水准为a=0.05,P≤0.05表示差异有统计学意义或有显著性差异。
研究结果
1.人工感染伤口所见：治疗后比较,臭氧水治疗组疗效远优于对照组以及洗必泰组,其中10mg／L与20mg／L臭氧水组疗效又优于5mg／L组。
2.白细胞总数变化：治疗3、6、9d后,10mg／L与20mg／L组白细胞总数无显著性差异(P>0.05),而前两者与5mg／L组在治疗后3、6d后有显著性差异(P<0.05)。治疗后9d,三种浓度的臭氧水治疗组白细胞的总数差异不显著。
3.光镜所见：治疗3d后,臭氧水治疗组炎症病理减轻,可见新生表皮细胞,以10mg／L组与20mg／L组明显；对照组炎症改变加重,出现肌纤维坏死。治疗6d后,洗必泰组炎症减轻不明显；臭氧水治疗组炎症病理进一步减轻,其中10mg／L组与20mg／L组上皮结构修复完整；对照组皮肤、肌肉坏死表现进一步加重。治疗后9d,洗必泰组炎症表现减轻,开始出现部分表皮细胞连接；臭氧水治疗组只有轻微的炎症表现,无渗出,10mg／L组与20mg／L组大部分恢复正常皮肤,肌纤维肉芽组织大量生长；对照组出现慢性炎症的表现。
4.EGF含量变化：在治疗各个阶段,治疗组与对照组比较以及洗必泰组与臭氧水治疗组比较均有显著性的差异(P<0.05),治疗3、6、9d后,10mg／L与20mg／L组无显著性差异(P>0.05),而前两者在治疗后3、6d后,EGF含量高于5mg／L组。而在治疗后9d,三种浓度的臭氧水治疗组EGF含量差异不显著。
5.TNF-α含量变化：在治疗各个阶段,治疗组与对照组比较以及洗必泰组与臭氧水治疗组比较均有显著性的差异,治疗3、6、9d后,10mg／L与20mg／L组无显著性差异,而前两者与5mg／L组在治疗后3、6d后有显著性差异,在治疗后第9d差异不显著,5mg／L组TNF-α含量高于前两者。
结论
1.中、高浓度臭氧水的抗炎消毒效果优于低浓度组,而中与高浓度组的效果没有明显差异,提示在临床应用中,10-20mg／L的臭氧水已足够控制甚至消灭创面感染。
2.EGF与大鼠创面的抗炎修复结局成正相关性,臭氧水治疗能促进创面组织中的EGF含量的升高,而且与其浓度有关,其中以10mg／L与20mg／L浓度组最佳,EGF含量在治疗过程中逐步增加。
3.臭氧水治疗对创面中TNF-α含量影响异于EGF,臭氧水治疗组中TNF-α含量普遍低于洗必泰组以及对照组,其中10 mg／L与20 mg／L浓度臭氧水治疗组中的TNF-α含量最低。臭氧水在治疗感染性创面的过程中降低了TNF-α含量,从而抑制了NF-KB系统的活性,达到抗炎修复组织的目的。
Background
Ozone (O3) with strong oxidation and instability, in the air at normal temperature and pressure easily reduce to oxygen (has half-life about 45 minutes). Ozone can soluble in water, in the air at normal temperature and pressure,it's solubility in water is 13 times higher than oxygen,and 25 times higher than air.However,the speed of decomposition in water is much faster than that in the air.For example,if the concen-tration of ozone in water is 6.25×10-5 mol/L(3mg/l), the half-decay period is 5～30min,but it decompose slowly in purified water, the half-decay period is 20min in distilled water or tap water.When in the redistilled water,only 10%of ozone decompose after 85 min,if the temperature of the water close to 0℃,ozone will be more stability.
Medical ozone (O2-O3) is the mixture of ozone and pure oxygen,which can quickly kill bacterium under oxidation-reduction reaction,it's consider as one kind of broad and utility bactericide duo to its stronge oxygenation ability. Along with recognizant and lucubrate oxygen-ozone to characteristic of biopharmacy, oxygen-ozone gradually expand to midecal area. In Europe, medical ozone are widely used for the treatment of diabetic foot wide, trauma and the healing of skin ulcers, stroke, ulcerative colitis, arthritis, prolapse of intervertebral disc, et al, its clinical efficacy has been fully affirmed.Ozone water, solubled in water by ozone gas,is more stronger in sterilizing, decomposition and promotion, organics and heavy metalcan be break dowm or transform into harmless material.Considering the character of ozone water, it is widely used in debrideing infected wound,mouthwash, vagina washing by hakeem domestic and overseas,and gain wonderful effect.On the other side,the facilities of prepare ozone water are small, the process is simple and quick,it couble bo conservation in the frige for 3-4days,all of which make ozone water much superior than traditional antiseptics.
Clinical results show that ozone therapy has significant advantages, but why it has not really promoted, and even be refused by many doctors and patients? The most fundamental reason is that the basic research of ozone therapy is lagging far behind in clinical practice, many roles and mechanisms of the therapy still unknown, what limit its expansion of the field of clinical treatment. In particular, the lack of basic research on safety of the ozone therapy blocks its development. So the safety of medical ozone therapy studies appears to be every important.
At present, the related research focuses on the ozone in the blood cells, in particular the impact of erythrocytes. Ozone has a strong sense of oxidation, contact a certain concentration and duration of ozone will cause the body damage. Most research on the ozone focus on the affection on blood erythrocytes.
Although plasma has series of buffer system, such as antioxidant enzymes and glutathione, to prevent accumulation of the superoxide anion and hydrogen peroxide, which may produce alterations of tissue structure secondary to cell membrane degradation, but there is still part of the ozone in the plasma generates reactive oxygen species (ROS). In vitro experiments, the ROS can trigger a number of biochemical pathways. ROS degradation in 0.5 to 1.0 minutes by antioxidant system in the plasma. As soon as contact and dissolved in the plasma, ozone react with anti and polyunsaturated fatty acids, and cascade generated a series of complexes which had been definited, such as hydrogen peroxide and lipid peroxidation products. Studies by Bocci, etc, suggested that when concentration of medical ozone was less than 80μg/ml, buffer systems and anti-oxidation system in the blood and red blood cell, can resistant the ozone oxidization and avoid cells and tissue damage, thus ozone can play a role as drug. When ozone concentrations more than 8μg/ml, it causes tissue damage easily, with the concentration of ozone increasing, there is more obvious damage.
However, the past reseashs are mainly concentrate on the anti-inflammatory, analgesia and immunoloregulation,the fewer studies on ozone water are localize on clinic.For this reason,it's necessary to find out,compare to ozone water,does different density of ozone water has different effert? What is the transmutation of organism contribute to ozone water,and what is the mechanicsm? Clinical physicians use ozone for treatments common concern these questions and the latter affect patients on the acceptability of the ozone treatment in a certain extent, and block the development of ozone therapy. But we have not yet seen relevant reports.
Purpose
1.To study the healing effect of different density of ozone water on the infected wound;
2.To explore the changes in endogenous growth factors and their relation with wound healing by ozone water.
Method
1. First part:The role of ozone water on antiinflammatory and reparation of infected wound.
50 vista rats were randomly divided into five groups:chlorhexidine treatment group、5mg/L、10mg/L、20mg/L ozone water treatment group and contrast group(without any special treatment). The back of rats were artificially formed the infected wound. After 48 hours of infection, the chlorhexidine or ozone water was given, once a day,for 9 times. Before and after the 3rd,6th,9th day of treatment, the alteration of infected wound was observed. The WBC of tail venous blood was detected and the incised skin and musle tissue was lstopathologically analysed by light microscope.
2. Second part:The changes in endogenous growth factors and their relation with wound healing by ozone water
Based on the first part of the experiment, before and after the 3rd,6th,9th day of treatment, harvested the muscle and skin tissue samples of the wound.The amounts of endogenous epidermal growth factor(EGF) and tumor necrosis factor-α(TNF-α) in infected wound were determined and their relation with the wound healing by ozone water were studied.
3. Statistics analysis:The experimental data was analyzed by the software of SPSS 13.0, and an associated probability of 5%(P<0.05) was considered significant.
Results
1. Artificial infected woud findings:Compare with post-treatment, ozone water have the better effect to promote the healing of infected wound, especially the result of 10mg/L and 20mg/L are better.
2. Total white blood cell changes:After the 3rd,6th,9th day of treatment, no significant difference in the total number of white blood cell betweem 10mg/L and 20mg/L ozone water treatment group (P> 0.05).But the first two have significant difference with 5mg/L ozone water treatment group after the 3rd,6th day of treatment.After the 9th day of treatment, the total number of white blood cells was no significant difference amon three kinds of concentrations of ozone water treatment group.
3. Light microscopy findings:After the 3th day of treatment, Ozone water treatment group reduce inflammation in pathological, newborn epidermal cells can be seen, especially in the result of 10mg/L and 20mg/L groud; Inflammatory changes in the contrast group was increased, muscle fiber necrosis can be seen. After the 3th day of treatment,reduce inflammation in pathological of chlorhexidine treatment groud was not obvious, the inflammatory pathology of ozone water treatment group were further reduced. Epithelial repair integritily in the 10mg/L and 20mg/L groud; Skin, muscle necrosis increased in the contrast group. After the 9th day of treatment, inflammation in pathological of chlorhexidine treatment groud was reduced, Some epidermal cells appeard to connect; Ozone water treatment group have only mild inflammation and no effusion, 10mg/L and 20mg/L groud mostly return to normal skin, The growth of granulation tissue and muscle fibers were significant, The contrast group appeared to chronic inflammation.
4.Content changes of EGF:In all stages of treatment, the treatment group compared with the control group and the chlorhexidine group compared with the ozone water treatment group,there were significantly difference (P<0.05). After the 3rd,6th and the 9th day of treatment,ther was no significant difference betweem 10mg/L and 20mg/L groud (P> 0.05).But the amounts of EGF in the first two were much greater than those in 5mg/L groud after the 3rd,6th day of treatment. After the 9th day of treatment, the Content changes of EGF were no significant difference amon three kinds of concentrations of ozone water treatment group.
5. Content changes of TNF-α:In all stages of treatment, the treatment group compared with the control group and the chlorhexidine group compared with the ozone water treatment group,there were significantly difference (P<0.05). After the 3rd,6th and the 9th day of treatment,ther was no significant difference betweem 10mg/L and 20mg/L groud (P> 0.05).But the amounts of EGF in the first two were significant different from 5mg/L groud,5mg/L groud was higher.
Conclusion
1. Medium and high concentration of ozone water groud have the better effect to promote the healing of infected wound than the low one,and the first have similar effect,which indicated that 10-20mg/L ozone water is Enough to control and even eradication of wound infection.
2. There is a positive correlation betweem EGF and wound repairment in rats,ozone water treatment can increase the content of EGF,and have a relationship with its concentration,10mg/L and 20mg/L groud have the best effect,the content of EGF gradually increased during the treatment process.
3. Opposite to the EGF,the content of TNF-αin ozone water treatment groud was lower than that in the chlorhexidine treatment group and contrast group,10mg/L and 20mg/L groud were the lowest. Ozone water treatment can reduce the content of TNF-α, thereby inhibiting the activity of the NF-κB system,finaly reach the purpose of antiinflammatory and reparation.
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