Yu-Lian Yin, Tian Meng, Li-Na Ma, Yi-Wei Fan, Yi-Fan Cheng, Yuan-Yuan Zhong,Hong-Feng Chen

Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China

Keywords:Chronic refractory wound Jiuyi powder Macrophage Phenotypic polarization Rats

ABSTRACT Objective: To observe the effect of Jiuyi powder and its active components on the bacterial culture and macrophage phenotypic factors of the chronic refractory wound rat model, and to explore its mechanism of removing decay and promoting muscle growth. Methods: SD rats were divided into control group, Jiuyi powder group, Shengdan group, and calcined gypsum group, with 8 rats in each group. MRSA-infected skin lesions and wounds were used to build a model of chronic and difficult-to-heal wounds in rats. After the model was formed, the control group was treated with daily routine nitrofural disinfection and replaced with sterile gauze. On the basis of the control group, quantitative Jiuyi powder, Shengdan powder, and calcined gypsum powder were used for dressing change, once a day for 7 consecutive days.Before and after the last administration, collect rat wound secretions for bacterial culture,inducible monoxide nitrogen synthase content. At the same time, after the last administration,the rat wound tissue was excised for histopathology and immunofluorescence double staining to label macrophages and their M1 phenotype. Results: After the last dressing change, the wound healing of Jiuyi powder group was better than the other groups, and the wound healing rate of each group had significant difference (P ＜0.05). The histomology showed that the inflammation of Jiuyi powder group was controlled and had a healing trend.After the last drug change, the contents of TNF-α, IL-6 and iNOS in serum of all groups decreased, and the contents of IL-6, TNF-α and iNOS in serum of Jiuyi powder group decreased significantly before and after medication (P＜0.05).There was statistical significance in serum IL-6 content between calcined gypsum group and Shengdan group before and after medication(P＜0.05).In addition, the results showed that the contents of IL-6 and iNOS in serum of Jiuyi powder group were statistically different from those of the control group (P＜0.05).Tissue immunofluorescence double staining showed that the positive rate of M1 macrophages in Jiuyi powder group and Shengdan group was significantly lower than that in control group (P＜ 0.05).The MRSA negative conversion rate of Jiuyi powder group and Shengdan group was better than that of the control group and calcined gypsum group (P＜0.05). Conclusion: Jiuyi powder can improve the inflammation of chronic refractory wounds, and has antibacterial, anticorrosion and myogenic effects. Its mechanism may be related to the inhibition of macrophage M1 phenotype polarization.

1. Introduction

Chronic refractory sores are a common clinical problem and a major challenge in surgical practice, characterised by an overall prolonged course and complex etiology, as well as a high degree of difficulty in healing, which can place a high medical burden on patients. The persistence of chronic refractory sores decreases the quality of life of patients[1] and it is for this reason that there is an urgent need to investigate the pathogenesis, the potential important targets and the search for appropriate treatments. Studies have shown that bacterial infections are one of the main factors affecting the healing of sores [2], with the main types covering Pseudomonas aeruginosa and Staphylococcus aureus [3]. Multi-drug resistant superbugs then cause the tissue to lock in a high inflammatory state and make healing difficult [4], which poses a great challenge for clinical treatment [5, 6]. To address this challenge, Jiuyi powder,one of the classical topical medicines in Chinese medicine, has remarkable effects in removing decay and regenerating muscle.The main components of Jiuyi powder are Sheng Dan (mercuric oxide) and calcined gypsum (calcium sulphate), which were found to be able to alter the local macrophages on the sore surface in the tissue of non-lactating mastitis patients. Macrophages are a common cell population with a highly heterogeneous and plastic overall functional state[7] , and their functional phenotype varies greatly depending on the local microenvironment, performing various functions to guide the physiopathological aspects. During the healing process, macrophages initially accelerate their phagocytic function due to local inflammatory stimulation of the sore, actively removing necrotic tissues and clearing invading pathogenic microorganisms, and when the inflammation is largely controlled,macrophages polarize into another functional phenotype that can secrete different cytokines, which facilitate subsequent cellular repair and promote cell proliferation, playing a pro-healing role in the process of vascular and granulation renewal. This substance facilitates subsequent cellular repair, as well as promoting cell proliferation and acting as a healing agent in the process of vascular and granulomatous regeneration. Macrophages have been reported to play an important role in the clearance of Staphylococcus aureus in infected mice [8], and differences in local macrophage content and phenotypic polarization during sore healing will affect the antiinflammatory and repair program of the sore [9].

In this thesis, a chronic refractory sore model was established in rats using a methicillin-resistant Staphylococcus aureus (MRSA)strain to model skin lesions and to observe the efficacy of the active fraction of Jiuyi powder on chronic refractory sores. The morphology of the sores, the content of M1 macrophage phenotypespecific factors and the immunofluorescence double staining were measured to investigate the efficacy and mechanism of action of Jiuyi powder in the treatment of chronic refractory sores caused by MRSA infection.

2. Materials and methods

2.1 Animals

A total of 32 SD rats, each weighing an average of (200±20) g,were purchased from the Experimental Animal Research Centre under the Shanghai University of Traditional Chinese Medicine.Prior to the start of the experiment, the rats were firstly acclimatized and fed for 7 days, with each unit of 2 rats being housed directly inside a plastic cage with 2. 5 cm wood shavings bedding, while ensuring that the circadian rhythm of the mice was 12 h. The ambient temperature was set at 18-24 C and the relative humidity was maintained at 50%-70% during the feeding process. The entire study was approved by the Experimental Animal Welfare and Ethics Committee of Shanghai University of Traditional Chinese Medicine(PZSHUTCM191018006).

2.2 Drugs

The Jiuyi powder, Sheng Dan and Calcined Gypsum used in the experiments were all purchased from the Traditional Chinese Medicine Preparation Laboratory of Longhua Hospital, Shanghai University of Traditional Chinese Medicine, and were also registered accordingly.

2.3 Key reagents and instruments

IL-6 (Interleukin-6), iNOS (Inducible Nitric Oxide Synthase),TNF-alpha (Tumor Necrosis Factor-alpha) rat ELISA All kits were purchased from Shanghai Jianglai Biotechnology Co., Ltd. with lot numbers JL21441, JL13202 and JL20896 respectively; CD11C antibody was purchased from Bioss with lot number Bs-2508R;F4/80 antibody, Goat anti-Rat and Goat anti-Rabbit were purchased from Abcam with lot numbers Ab16911, Ab150157, Ab150080; key instruments: tissue slicer, TB-718 series automatic biological tissue embedding machine, RE52CS1 rotary evaporator and Neofuge 13R cryogenic centrifuge, etc.

2.4 Modelling method and grouping

The chronic refractory sores of rats were modelled according to the literature [10]. 4% chloral hydrate (1mL/100g) was injected into the peritoneal cavity of the rats. A depth of fascial layer was required.The sores were sprayed with MRSA and covered with gauze to ensure a good fit between the skin sutures and then secured with adhesive tape. 24 h later, the corresponding sore models were made.The experimental animals were classified using the random number table method and divided into control group, Jiuyi powder group,Calcined Gypsum group and Sheng Dan group, with a total of 8 animals in each group.

2.5 Intervention method

After successful modelling, the control group was covered with sterile gauze and bandaged after routine disinfection of the sore surface. The composition of Jiuyi powder mainly consisted of forged plaster (calcium sulphate) and Sheng Dan (i.e. mercuric oxide) with forged plaster (calcium sulphate) in the ratio of 1:9. The specific dosage was firstly referred to the dose of 1.5 mg/cm2 specified in the safety and normative protocol for topical application of Jiuyi powder[11], and the body surface area between human and rat was also included for conversion, and the final equivalent dose corresponded to 9.255 mg/cm2. The final equivalent dose corresponded to 9.255 mg/cm2 , therefore the Jiuyi powder group was evenly distributed on the sore surface on top of the control group. In the calcined plaster group, 9 parts of calcined plaster were taken according to the dose used in the Jiuyi powder group, and in the Sheng Dan group,1 part of Sheng Dan was taken according to the dose used in the Jiuyi powder group. The sore was changed daily and the size of the sore surface and the condition of the granulation, pus decay and epithelium on it were recorded before, on the first day after, on the third day after, on the fifth day after and on the seventh day after the change of medicine.

2.6 Testing indicators and methods

2.6.1 Sore surface healingThe sore surface was photographed by camera on the first day of successful moulding and on days 1, 3 and 7 after dressing change,and then the sore surface healing rate was calculated at a later stage by the formula: (size of sore surface on initial day - size of sore surface on selection day)/size of sore surface on initial day × 100%.

2.6.2 Histomorphology

The histomorphology of the sore surface is observed, then the specimen is selected, embedded, fixed and sectioned, and the different histomorphologies are observed using a light microscope.

2.6.3 Elisa methodAortic blood from the abdomen of rats is selected, centrifuged, and the serum is separated to obtain the appropriate serum.

2.6.4 Immunofluorescence staining

Each tissue was taken, dehydrated, transparent, wax dipped,embedded and sectioned; drops of F4/80 and CD11c antibodies were prepared at 1:50 and 1:200 ratios respectively and left overnight at 4 C. Subsequent preparations were made at a ratio of 1:200, followed by dropwise addition of and secondary antibody working solution in which incubation was carried out at 37 C for 2 hours. After baking the slices, anti-fluorescence burst sealer was applied to seal the slices. Acquisitions were made at 100x and 400x field of view respectively; ImageJ software was used to count the fluorescence optical density of the yellow portion under the field of view.

2.6.5 Microbiological culture of bacteria on the sore surface

Samples were taken before and 7 d after the dressing change using disposable sterile swabs and sent to PCR for microbiological detection.

2.7 Statistical methods

SPSS 20. 0 software was used to analyse the data. x±s was used to express the results of measurement data, and x±s was used to compare between groups.

3. Results

3.1 General condition of the rats and healing of the sore surface

After 7 days of observation, 2 rats died in the Shengdan group,while all other groups were in good mental condition and had no abnormal food intake. The healing rate of the sores in the Jiuyi powder, Sheng Dan and Calcined Gypsum groups was better than that in the control group, with the Jiuyi powder group having the best healing situation. On day 3, there was no significant difference in the size of the sores between the groups (F=1.931, P=0.1475);on day 7, there was a significant difference in the healing rate of the sores between the groups (F=17.04, P＜0.001), with the Jiuyi powder group healing better than the other groups, Table 1.

3.2 Histomorphological observation of the sore surface of the rats in each group

The histological observation and HE staining results of the sore surface of rats in each group showed that after the last change of medicine, the neutrophils in the control group were densely distributed, suggesting heavy inflammation, while the neutrophils in the Jiuyi powder and Sheng Dan groups were loosely distributed and there were more fibrous tissues in the Jiuyi powder group,suggesting that the inflammation was controlled and had a tendency to heal, see Figure 1.

Figure 1 HE staining results (400x) in the control (DZ), Jiuyi powder (JYD),Calcined Gypsum (DSG) and Sheng Dan (SD) groups after the final drug change

Table 1 Healing of sore surface in rats in each group (unit: cm2)

3.3 Comparison of macrophage phenotypic factor content

After successful moulding, there was no statistically significant difference between groups in the levels of macrophage M1-specific markers IL-6, iNOS and TNF-α in the serum of each group before the drug change (IL-6: F=1.481, P=0.2552; TNF-α::F=0.3611,P=0.7819; iNOS: F=1.527, P=0.2436). After the final drug change,the serum levels of TNF-α, IL-6 and iNOS in all groups showed a decreasing trend, suggesting that the inflammation was controlled.After statistical analysis, it was found that the serum IL-6, TNF-α and iNOS levels decreased significantly in the Jiuyi powder group after the drug change (all P ＜ 0.05); while the intra-group comparison of serum TNF-α levels only before and after the drug change was statistically significant in the control group (P = 0.0219);the intra-group comparison of serum IL-6 levels only before and after the drug change was statistically significant in the calcined gypsum group and the Sheng Dan group (P =0.0307 in the Calcined Stone Group and P=0.0148 in the Shengdan Group); in addition,there were also differences in the changes of serum specific markers between the groups after the drug change, and the results showed that the serum levels of IL-6 and iNOS in the Jiuyi powder Group were statistically different from those in the control group after the drug change (IL-6: P=0.0148, iNOS: P=0.021); the serum levels of TNF-α in the Calcined Stone Group were statistically different from those in the Jiuyi powder Group (P=0.0148). There was a statistically significant difference (P=0.0213) and the difference in iNOS content in serum between the Sheng Dan group and the Jiuyi powder group (P=0.0493), Figure 2-4.

Figure 2 IL-6 content in each group before and after dressing change

Figure 3 TNF-α content in each group before and after dressing change

Figure 4 iNOS content in each group before and after dressing changeNote:1 represents before dressing change,2 represents after dressing change

3.4 Immunofluorescence staining results

After the final drug change, by immunofluorescence staining,bright green corresponds to the F4/80 expression site, bright red corresponds to the CD11c expression site, blue corresponds to the nuclear staining expression, and yellow corresponds to the labelled completed M1 macrophages. Statistically, the ANOVA between groups F=7.221, P value was 0.0004, and the fluorescence optical density analysis of labelled M1 macrophages was significantly different between groups, including the control group and the Jiuyi powder group (P was 0.0039) and the Sheng Dan group (P was 0.0474), and the difference between the 2 groups was statistically significant, Figure 5 and 6.

Figure 5 Immunofluorescence staining results for each group

Figure 6 Comparison of the fluorescence optical density of the immunofluorescence stained IF in each group

3.5 Microbiological culture results of sore surface bacteria

The first results of sampling after successful moulding in all groups were suggestive of MRSA infection. After the change of medication intervention, the sore surface secretions were re-collected for bacterial culture and the results showed that 5 cases were converted to negative in the Jiuyi powder group, 5 cases were converted to negative in the Sheng Dan group, 1 case was converted to negative in the Calcined Gypsum group and no cases were converted to negative in the control group.

4. Discussion

Macrophages play a key role in wound repair, as they not only remove and engulf microorganisms and necrotic tissues, but also secrete corresponding cytokines, which can be used to target cells for repair, effectively stimulate cell proliferation, and facilitate vascularization and granulation itself. At the same time, the different phenotypes of macrophages have many different physiological functions. The current macrophage types cover two main forms,corresponding to the M1 type, the classically activated macrophage,and the M2 type, the selectively activated macrophage [12]. During the inflammatory phase, M1-type macrophages can directly secrete TNF-α, IL-6, etc., and these will participate in the subsequent inflammatory response. After the onset of inflammation they are converted to M2 type and secrete anti-inflammatory factors such as Arg-1 and CD206, which are involved in tissue repair and wound healing [13-15]. Recently, several studies have found that Chinese medicine intervenes in the phenotypic changes of macrophages on the sore surface and has a positive effect on sore healing. Liu Chen [9]analysed the whole process of wound healing in diabetic rats. From the initial stage, the overall number of macrophages was relatively low, but in the later stage of healing, they continued to be expressed on the wound surface, and the expression of various cytokines associated with their phenotype also showed dynamic changes,suggesting that macrophages may be associated with diabetic wound healing. During their study, Meng Yujiao [16] et al. showed that the results of the blood activation and blood circulation formula and the benefit of Qi and warmth formula were beneficial to the actual healing of chronic skin sores in mice, promoting the development of sores from the inflammatory phase to the proliferative phase. Lin Yan[17] also reviewed the role of macrophage cell burial and phenotypic transformation in the improvement of chronic skin ulcers.

Shengdan is a good remedy for the treatment of "carbuncles and gangrene" in surgery. It is recorded in the book "Surgical Authentic- Sheng Bai Ling Medicine": "Whenever a sore does not close for a long time, use this to mix a little with fine research, and the mouth will be easily finished. This means that if a sore does not close for a long period of time, the use of a small amount of this powder mixed in with all the astringent medicines can have a miraculous effect.This is why it was said in the past that red and white pills were always used in surgery. The mercury preparation itself is corrosive and toxic, so it is necessary to add excipients to it in the course of clinical application, and to mix the topical Sheng Dan preparations in proportion, choosing the right drug according to the proportion of pus and rot. The overall efficacy of Jiuyi powder is relatively good, as well as being simple to handle and relatively inexpensive,hence its current use is very widespread. Current research results show [18-19] that the pharmacological effects of Shengdan itself mainly include: (1) bactericidal effect: the HgO inside Shengdan can be reduced to obtain Hg2+, which then combines with each other and the sulfhydryl groups inside the bacterial respiratory enzymes,a condition that causes bacterial death. (2) Antiseptic effect: the mercuric nitrate is a soluble salt that decomposes when added to water to obtain an acidic solution, which has a corrosive effect and is able to necrotize proteins in diseased tissues and separate them from healthy tissues. (3) Myogenic effect: Jiuyi powder can stimulate capillary production and expansion in the granulation of the wound, reducing microthrombosis and facilitating wound healing. In our group, Jiuyi powder was used in small doses to treat chronic refractory sores on the breast, and has achieved definite clinical efficacy [19-21], showing that Jiuyi powder has a good effect of removing decay and regenerating muscle. However, no previous studies have been conducted on the mechanism of action of Jiuyi powder, and the findings in this section are therefore complementary to them. In addition, in this study, it was observed for the first time that Jiuyi powder could inhibit the secretion of TNF from macrophages in inflammatory conditions and reduce the production of IL-6, iNOS and other inflammatory substances.Immunofluorescence staining also suggested that Jiuyi powder could reduce M1 macrophage polarization. At the same time, the Jiuyi powder group was superior to the control group and the calcined gypsum group in terms of decay and healing of the sore surface;although the ShengDan group showed a better decaying effect, the rat mortality rate in the group was higher and its safety was poor.Therefore, in conclusion, Jiuyi powder's Sheng Dan component can play a good role in lifting pus and eliminating decay, while the combination with calcined gypsum can dilute the concentration of Sheng Dan and reduce the local toxic side effects. The classical combination of Jiuyi powder is complementary to each other and can play a role in reducing the toxicity and stabilizing the effect.

In conclusion, this study found that Jiuyi powder and its effective components could regulate the M1 phenotype of macrophages and exorcise decay and regenerate muscle on inflammatory sores through animal experiments. It was also found that the ratio of Jiuyi powder could ensure its effectiveness on the basis of safety. It is also the first time to investigate the potential action links and targets of Jiuyi powder and its effective components from the perspective of macrophage phenotypic polarization, and more in-depth studies are planned in the future to supplement the theoretical basis of Jiuyi powder, a classical traditional Chinese medicine topical agent, which will be beneficial to the promotion of Jiuyi powder in the clinical field of surgery.

Conflict of interest

All authors declare that they have no conflict of interest.

Authors' contribution Yulian Yin: proposed the idea and wrote the paper; Yulian Yin,Meng Hata, Lina Ma, Yiwei Fan, Yifan Cheng and Zhongyi Yuan:completed the relevant experiments; Hongfeng Chen: overall control and review of the paper.