Qiang Han, Guo-Bin Liu, Shi-Meng Yan, Ren-Yan Huang, Feng Xu, Xiao-Ming Hu, Chen-Yan Shi, Xiao Yang, Li-Xiang Wang, Shao-Qing Yan

Shuguang hospital affiliated to Shanghai university of traditional Chinese medicine, Shanghai university of traditional Chinese medicine

Keywords:

ABSTRACT

1. Introduction

As a complex chronic disease, diabetes has become a global public health problem.Some studies suggest that 420 million people worldwide are currently living with diabetes, with the number expected to reach 640 million by 2040.[1] According to the statistical results in 2020, there are 34.2 million people with Diabetes in the United States, accounting for 10.5% of the population (National Diabetes Statistics Report, 2020).China's diabetes data is also not optimistic, it is estimated that there are about 110 million patients with diabetes in China, the number of prediabetic about 490 million.[2]Diabetic foot ulcer is an important risk factor for amputation in diabetic patients, and more than 15% of diabetic foot ulcer patients experience amputation.[3][4][5]All kinds of pathological changes resulting from diabetic status, including reduced wound repair cells and growth factor activity, persistent chronic inflammation, reduced peripheral blood flow and reduced local angiogenesis, may lead to the difficulty in healing diabetic foot ulcers.[6][7]

Diabetic foot ulcer is a serious lower extremity complication of diabetes, which usually leads to amputation at a later stage. The etiology of diabetic foot ulcer includes many factors. Persistent inflammation and blocked proliferation of tissue cells are the important factors leading to the difficulty in healing of diabetic foot ulcer. Although the treatment methods are constantly updated, the treatment of diabetic foot ulcer is still a clinical problem.

Wound healing is a complex process that requires the precise coordination of all kinds of cells and biomolecules to achieve wound tissue repair. The process of wound healing is related to inflammation, cell proliferation, angiogenesis, and tissue formation.[8]Delayed wound healing is the most common problem in diabetic foot ulcers. The inflammatory response in the healing process of diabetic foot ulcers results in the disorder of the coordination of various cells and cytokines, which is further disrupted by bacterial colonization or infection.[9]The mechanism of wound healing is complex. One of the important reasons is the persistent inflammatory response and the delay in cell proliferation and remodeling.[10]Studies have shown that wound healing of diabetic ulcer is associated with increased pro-inflammatory cytokines such as il-1, il-6, and TNF- radiation.[12][13][14]Some clinical studies have also shown impaired cell proliferation, reduced angiogenesis, and reduced growth factors in diabetic patients.[9][13]

Diabetic foot ulcer is a chronic refractory ulcer. Many external Chinese medicine preparations have good antibacterial and bacteriostatic effects and can significantly promote the healing of diabetic ulcer.[14][15][16]

Zizhu ointment is an effective prescription for treating diabetic foot ulcer in our hospital. We found that zizhu ointment could improve the cure rate of chronic skin ulcer of diabetic foot and lower limbs in the previous clinical case study.[17][18] In this study by observing the purple zhu ointment effect on healing of diabetic foot ulcer rats to explore the treatment mechanism, the results show that the purple zhu ointment can improve diabetic foot ulcer wound inflammation, improve wound healing, suggest it can be used as a new clinical application in diabetic wound healing wound dressings, diabetic foot ulcer of diabetes for purple zhu ointment treatment to provide experimental basis.

2. Materials and methods

2.1.1 Experimental animals:

Male Sprague - Dawley (SD) rat, clean grade, body mass (180 ± 10) g, provided by laboratory animal center, Shanghai university of traditional Chinese medicine.Feeding environment: room temperature (232) ℃, relative humidity (5610) %, 12 hours of no light /12 hours of artificial light, free diet.The experimental procedures follow the guidelines of the laboratory animal center of Shanghai university of traditional Chinese medicine.

2.1.2 Main reagents and drugs

Streptozotocin (STZ), Sigma;Preparation method of STZ solution: citric acid (FW: 210.14) 2.1g was added to 100mL double distilled water to form solution A. Sodium citrate (FW:294.10) 2.94g was added to 100mL double distilled water to form liquid B. When used, A and B were mixed in A proportion of 1:1, and PH value was determined by PH meter to be 4.2-4.5, that is, the required STZ citric acid buffer. At the time of injection, the solution was dissolved in citric acid buffer solution at a concentration of 1%, and STZ solution of 35mg/kg was injected according to the fasting body weight. The injection was completed within 30 minutes after preparation.

High fat feed ingredients: basic feed 73% (barley 15%, wheat 31%, corn 15%, bran 15%, fish meal 6%, soybean meal, oil and salt 18%), lard 20%, milk powder 2%, cholesterol 1%, sucrose 4%, provided by laboratory animal center of Shanghai university of traditional Chinese medicine.

Quality control strain staphylococcus aureus - ATCC28923, escherichia coli - ATCC25922, provided by Shanghai center for disease control and prevention;Recombinant bovine basic fibroblast growth factor for external use (trade name: berfuji, rb-bfgf), zhuhai yicheng biological pharmaceutical co., LTD., national drug approval S10980077.

The main ingredients of zizhu ointment include vermilion, combia, blood, astragalus, ejiao and borneol. All the drugs were purchased from Shanghai kangqiao traditional Chinese medicine yinpian co., LTD., and passed the quality control standard.According to the configuration process by wuhan mayinglong workshop processing and production.

The ratio of the main medicine to the base material was 1:8, in which the main medicine (by weight) was: cinerea cinerea, radix astragali, radix astragali, borneol. The ratio of each drug was 7:3:3:6:5:1. Excipients and ratio: poloxam, poloxam, water, glycerol, PEG, PEG 1500, PEG 4000, ethyl p-hydroxybenzoate. The proportion of each auxiliary material shall be 34:24:70: 26:9:3:06.

2.1.3 Main instruments

Scald meter sql-5q scald meter, Shanghai xin soft information technology co., LTD. ELISA kit, BD company.PureLink RNA Mini Kit (Thermo fisher) was used to extract mRNA.SuperScript III III first chain synthesis systems, Life Technologies, inc.SYBR ? GreenFastMix, Takara inc.Blood glucose test paper, roche. Mercury meter F 1 732, Shanghai huaguang instrument co., LTD.

2.2 Establishment of diabetes model

In this study, diabetes was established by using high-fat diet combined with STZ drug, ulcer wound was prepared by constant temperature and constant pressure scald apparatus, and a diabetic foot ulcer model was established, that is, the experimental model. The non-diabetic ulcer model and the diabetic model were used as the control model for the control model.[19]The specific operation method is as follows.

2.2.1 Diabetes preparation stage method:

Experimental group model (C, D, Z0, Z1, Z2, Z3) and non-diabetic ulcer model (B). After 8 weeks of high-fat feed feeding, fasting for 24 hours, 1.5% streptozotocin was dissolved in citric acid - sodium citrate buffer at a pH value of 4.5 and injected into the left lower abdomen at a dose of 35 mg/kg. Evaluation criteria:After 24h of injection, the blood glucose was greater than 16.7mml /L, resulting in excessive drinking and urination, back hair contamination, and wet bedding. After 3d testing, the blood glucose was still greater than 16.7mml /L, which was considered as diabetes, and then the blood glucose was changed to normal feed. Non-diabetic ulcer model (A) rats were fed normal diet for 8 weeks, fasted for 24 h, and injected with an equivalent amount of citric acid - sodium citrate buffer.

2.2.2 Diabetes ulcer model preparation phase

Preparation after 1 week, experimental model (C, D, Z0, Z1, Z2, Z3) and reference model (A, B), with 3% pentobarbital sodium after intraperitoneal injection of 2 mL/Kg dose of anesthesia, use SQL to 5 q constant temperature constant pressure burns instrument, set the temperature of 90 ℃, the contact time of 10 s, pressure is 9.8 N, choose area of 4 cm probe, back after anesthesia bottom skin burns.2Evaluation criteria: paleness, swelling or blisters were observed immediately at the scald site. Border area 24 h after the skin pathological section, HE staining observation on depth Ⅱ burns.

2.2.3 Animal grouping 48 SD rats were divided according to the random number table method

Control model treatment: non-diabetic ulcer group (A) used normal saline for daily dressing change; The diabetes group (B) was not treated as a blank control of diabetes. The experimental model group was divided into two groups according to the drug intervention: group D was treated with normal saline for daily dressing change, and group C was treated with western drug bfzil (recombinant bovine basic fibroblast growth factor, rb-bfgf) for 800IU daily dressing change.In the drug intervention group, Z0, Z1, Z2 and Z3 were treated with substrate (gauze containing 0.3125g substrate), low (1/2 times), medium (normal dose), and high (2 times) doses of zizhu ointment gauze, respectively.(the normal dose is 0.3125g of zizhu ointment per tablet, and the high and low doses are configured with the drug concentration doubled/decreased.Preparation method of medical gauze: make 4 medical gauze sheets of 20g ointment, cut each sheet into 16 pieces, and change the dressing one at a time.The samples were anesthetized after 14 days of observation.

2.3 Test method

HE staining: used for histological analysis of kidney and liver. After the mice were killed, fresh kidney and liver were collected and fixed in 4% paraformaldehyde. The paraffin sections were sectioned at the thickness of 8 meters and stained with H&E solution. Section pictures were taken and analyzed using a microscope.

Immunohistochemistry: wound tissue was harvested after the rats were euthanized with pentobarbital sodium. Then the wound tissue was fixed in 4% formaldehyde buffer solution for 24 hours. The sample was then subjected to standard dehydration for 24 hours at a series of increased ethanol concentrations. The tissue was then embedded in paraffin and sectioned using a histological slicer. Install the 8 foot-thick tissue slices on the glass slides and dry them on the heating plate overnight. The slides were then washed in deionized water and washed in PBS. Thermally induced antigen repair was performed. The slides were placed in Citrate 6 buffer (Sigma) at 94℃ for 20 min. The slides were then washed in PBS and permeated in a 0.2% triton-x-100 PBS solution for 10 minutes at room temperature. The sample was then sealed in 5% goat serum (microporous) in PBS for 1 hour at room temperature. The primary and secondary antibodies were diluted in a closed solution. The primary antibody CD34 + or VEGF was incubated overnight at 4℃ and incubated at room temperature for 1 hour in the secondary antibody (1:200). Samples were then taken in ABC kits (Vectorlabs) and DAB (Vectorlab) solutions according to the manufacturer's instructions.

Enzyme-linked immunosorbent assay (ELISA) : use commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA) to determine the concentrations of inflammatory cytokines (including IL6, CRP, and no-1) in wounds, according to manufacturer's instructions.Calculate the protein concentration from the standard curve. These values were normalized to the total protein content as determined by the Pierce BCA assay.Western-blot analysis of total protein in cleaved wound tissue in RIPA buffer (Sigma), including the complete/phosphatase termination mixture (Millipore).Protein concentrations were measured using Pierce BCA assay (Thermo). Then the lysates were separated by 6-10% sds-page (20 livg per lane) and transferred to the nitrocellulose membrane. Will transfer membrane with a closed solution [5% milk (BD) in TBST incubation for 1 hour at room temperature, and then with the first antibody incubation under 4 ℃ for the night. The film and HRP - 2 (Millipore) resistance of conjugated incubation for 1 hour at room temperature. Use ECL kit (Millipore) immune reactivity. Using ImageJ (NIH) analysis immune reactivity optical density in the band.RNA isolation and real-time PCR: according to the manufacturer's instructions, PureLink RNA Mini Kit (Thermo fisher) was used to separate total RNA from wound tissue. The first Strand cDNA was synthesized using the SuperScript III iii-strand Synthesis System from Life Technologies. SYBR Green was used as a real-time PCR indicator, and ABI StepOne Plus real-time PCR system was used. PCR was performed at 95℃ for 40 cycles for 15 seconds and at 60℃ for 1 minute. The fluorescence is read during the reaction, allowing continuous monitoring of the amount of PCR products. Gapdh mRNA was used as the standardized data for the internal control. The primers are as follows: VEGF, forward 5' -ctgctgcaatgatgaagccc-3, reverse 5' -tcccgaaaccctgagggag-3 '; E-cadherin, forward 5' -cgaaggcctaagcacaacagc-3 ', reverse 5' -cggtgtacacagcattccacg-3; GAPDH, forward 5' -tgctgagtatgtcgtggagt-3 ', reverse 5' -gtcttctgagtggcagtgat-3 '.

Mercury (Hg) cold atomic absorption method: Mercury vapor has a strong absorption effect on ultraviolet light with a wavelength of 253.7nm. After the sample is properly treated, various forms of mercury are converted into mercury ions, which are reduced to elemental mercury with stannous chloride, and then measured with a mercury meter. The mercury concentration is proportional to the absorption value.

Method of operation: 1. Sample treatment: put the sample into a quartz boat (covered with a small piece of filter paper) and put the quartz boat into a quartz tube of tubular electric furnace. One end of the quartz tube is connected with the oxygen cylinder, and the other end is connected with the absorption tube containing 20ml absorption liquid. The furnace temperature was controlled at 750±50℃ with oxygen (flow rate 2L/min), and the mercury in the sample was enriched in the absorption solution for determination. 2. Determination: accurately absorb a certain volume of absorption liquid (after combustion) into the mercury reaction bottle, add 2m1 30% tin chloride solution, immediately plug the bottle, start the circulating pump, and read the maximum absorption value.

2.4 Statistical analysis

In this study, x±s was used to represent the measurement data conforming to normal distribution, while median was used to represent the measurement data not conforming to normal distribution. Non-parametric Mann Whitney U was used to test the difference between the two groups.One Way ANOVA was used to analyze the measurement data of two groups with normal distribution and homogeneity of variance. T-test was used for comparison within the same group, and Student's t test was used for comparison between the two groups. Variance analysis was performed after correction of variance variance.Nonparametric tests are used for those who do not follow normal distribution.Results of P<0.05 were considered statistically significant (* P<0.05, ** P< 0.01, *** P< 0.001). Analyze and plot all the results using GraphPad Prism.

3. Results

3.1 Hgs safety of zizhu ointment and the influence of the expression of inflammatory factors in diabetic foot ulcer

Figure 1 Determination of Hg content by cold atomic absorption method and the results of ELISA for inflammatory factors in each group of rats

Table 1 Hg index results of rats in each group (x±s)

Table 2 results of inflammatory cytokines in each group by ELISA (x±s)

In order to determine whether zizhu ointment can improve the inflammatory response in diabetic foot ulcer rat model, we used normal saline and western medicine berfuji as the control drug of zizhu ointment to treat diabetic foot ulcer (D) rats for 2 weeks. During the two weeks of the treatment period, venous orbital blood was collected regularly for blood mercury (Hg) content determination. After the observation, serum and wound tissue were obtained for inflammation indicator determination (figure 1A).

Because of zi zhu ointment Chinese medicine ingredient purity is more than 98% of HgS cinnabar, is both the main drug ingredients, there may be a certain drug toxicity, we established a purple zhu ointment Hg detection method, therefore the full assessment of drug safety, first of all, the evaluation to H&E staining on the liver and kidney morphology observation, the results showed that in the liver and kidney, purple zhu ointment treatment of rat liver and kidney tissue without exception, and the control group no difference.[20]Blood Hg levels were measured on day 3, day 10, and day 14 after the experiment. In diabetic foot ulcer rats, an increase in blood Hg level was observed in the number of days of medication, and Hg level in group Z3 was higher than that in the other two groups.

Effect of zizhu ointment on diabetic foot ulcer model. Compared with previous studies, the expression of inflammatory factors CRP, il-6 and NO in diabetic foot ulcer rats was increased compared with normal ulcer control rats (A) or diabetic rats (B).[21][22]In addition to low doses of zizhu ointment, other doses of topical zizhu ointment and b-fgf treatment significantly reduced CRP, il-6 and NO levels in diabetic rats.

3.2 Effect of zizhu ointment on wound tissue angiogenesis

Figure 2 immunohistochemical results of angiogenic factors in each group

Table 3 results of vascular growth factor rt-pcr in each group(x±s )

Table 4 immunohistochemical determination results of vascular growth factor in each group (x±s)

Angiogenesis plays an important role in wound healing and is related to the expression of angiogenic factors such as CD34 and VEGF.[23][24]The angiogenesis and proliferation rate of wound tissue were measured by immunochemistry and rt-pc on CD34, VEGF and e-cadherin. The results showed that moderate (normal) dose of zizhu ointment increased the expression of CD34 and VEGF in diabetic foot ulcer rats.

In contrast, low-dose and high-dose diabetic foot ulcer rats showed an increasing trend. This is consistent with the results of drug screening optimization for zizhu ointment in the previous pre-drug research work.

The expression of VEGF and e-cadherin genes was measured by rtpcr. Compared with normal ulcer control rats (A) or diabetic rats (B), the expression of VEGF and e-cadherin genes in the wound tissue of diabetic foot ulcer rats (D) was decreased, and the expression of VEGF and e-cadherin could be upregulated by zezhu ointment.

3.3 Effect of zizhu ointment on inflammatory pathways

Table 5 WB test results of inflammatory pathways in each group ( x±s )

Some studies have reported that the higher inflammatory response in the healing process of diabetic foot ulcer is also an important reason for the delay of wound healing.[25]Therefore, this study examined the role of zizhu ointment in regulating the inflammatory pathway of diabetic foot ulcer by detecting the classical inflammatory pathways NF- B and JNK. The results showed that NF- B- or JNK signal expression was increased in diabetic foot ulcer (D) rats compared with diabetic model rats (B) or normal ulcer model rats (A). It indicated that zizhu ointment could significantly inhibit the expression of NF- B- or JNK pathway in wound tissue of diabetic foot ulcer, thereby reducing the intensity of wound inflammation and promoting wound healing.

4. Discussion

In this study, the results showed that zichu ointment could promote the healing of diabetic foot ulcer by reducing the level of inflammatory factors in diabetic foot ulcer rats, down-regulating the expression of proinflammatory genes NF- B and JNK signaling pathway, reducing wound inflammation, and increasing angiogenic factors CD34 and VEGF to stimulate wound angiogenesis. Wound healing is a complex biophysiological process involving cell proliferation, angiogenesis, inflammation, wound closure and remodeling.[26]Diabetic foot ulcer is a complex clinical problem, and its treatment methods include surgical debridement, enzyme debridement, various new wound dressings, various growth factors and stem cell therapy, etc., and the clinical treatment effect is still not ideal. Chinese medicine has distinct characteristics and good efficacy in the treatment of diabetic foot ulcer. Almost all medical institutions in China have various forms of Chinese medicine compound preparations for the treatment of various ulcers. Studies have shown that traditional Chinese medicine can promote wound healing, regulate immunity, fight oxidation and fight inflammation.[27]The ingredients of zizhu ointment in this study included cinnabar, combia sinensis, dried blood, astragalus membranaceus, ejiao and borneol. Its single ingredient can promote wound healing.[28][29][30]Previous clinical case studies showed that zizhu ointment could promote the healing of chronic skin ulcer.[18]On this basis, the cellular and molecular mechanisms of zizhu ointment in promoting ulcer healing were discussed.

Wound healing is a finely regulated process with sequential inertia and chronology, characterized by four distinct but overlapping stages: inflammation, proliferation, wound closure and remodeling.[31]Impaired wound healing is usually accompanied by an abnormal inflammatory response, reduced angiogenesis, and inadequate fibroblast proliferation.[32][33][34]Therefore, in this study, we investigated the effects of zizhu ointment on inflammatory response, angiogenesis and cell proliferation.

Adequate expression of proinflammatory mediators is required in the production of cytokines to recruit neutrophils and macrophages to remove wound bacteria and other contaminants.[35]However, the continued expression of these inflammatory factors can lead to a delay in the healing process. Optimal release of CRP, il-6, and NO promotes wound healing, and these markers not only act as mediators involved in cellular inflammation, but also play an important role in regulating cell proliferation, angiogenesis, and wound recovery.[36][37][38]In this study, CRP, IL6 and NO levels were significantly increased in diabetic foot ulcer rats, but not in normal ulcer rats. The results showed that the concentrations of all three markers were significantly reduced by different dose concentrations of zizhu ointment. Angiogenesis is an essential process in wound healing, and the most effective angiogenesis factor is vascular endothelial growth factor (VEGF).[39]CD34, on the other hand, is an endothelial antigen that has been used to measure microvascular vascular density as a direct marker of the degree of neovascularization.[40] Studies have shown that angiogenesis of skin ulcers in diabetic rats is increased by the VEGF pathway.[41]This is consistent with the results of this study. The upregulation of angiogenic factors VEGF and CD34 was also observed in the wound tissue of diabetic foot ulcer rats, indicating that zizhu ointment can accelerate wound healing by increasing the VEGF level.NF- B nuclear transposition and JNK phosphorylation are thought to play key roles in the inflammatory phase of wound healing.[42] [43]In order to further investigate the mechanism by which zichu ointment inhibits inflammatory response, the JNK phosphorylation and NF- B pathway in the wound tissue of diabetic foot ulcer were detected, and it was found that zichu ointment significantly inhibited the activation of the JNK and NF- B pathways in the diabetic foot ulcer model. Combined with the results of this study, zizhu ointment can promote wound healing by inhibiting the inflammatory response mediated by the JNK and NF- B signaling pathways and reducing the excessive inflammatory response of diabetic foot ulcers on the wound surface. This study proved that zizhu ointment could effectively promote wound healing in diabetic foot ulcer rats. The pathway may be the regulation and balance of inflammation, angiogenesis and tissue regeneration, providing a scientific basis for zizhu ointment to treat diabetic foot ulcer patients.