Platelet-rich Plasma (PRP) in the Treatment of Diabetic Foot Ulcers and its Regulation of Autophagy

Abstract

Objective: Through clinical trials, this study observes the therapeutic effect of platelet-rich plasma (platelet-rich plasma, PRP) on diabetic foot ulcers and explored the relationship between the relationship between PRP and autophagy. Methods: Thirty patients with diabetic foot ulcer who met the relevant criteria were randomly divided into PRP treatment group and control group. In the PRP treatment group, the formed PRP gel was coated and bandaged on the diabetic foot ulcer wound, and the PRP treatment was repeated on the seventh day. The control group was covered with normal saline sterile gauze. Observe the healing rate of the wound in 7 days, 14 days and 21 days, the pain in 5 consecutive days and the healing time of the wound after treatment, collect wound granulation tissues before and twenty-first days after treatment then detect the expression of autophagy-related proteins (LC-3, P62) and inflammatory factors (IL-6, IL-10) in diabetic foot ulcer wound to investigate the potential relationship between PRP treatment of diabetic foot ulcers and autophagy and inflammatory responses. Results: The wound healing rate of diabetic foot ulcer patients in the PRP treatment group was higher than that in the control group on the seventh, 14th and 21st days, the healing time (31.40 ± 4.47) was better than that in the control group (43.20 ± 5.03) days, and the pain improvement was better than that in the control group (P < .05). The results of Western blot analysis and quantitative PCR of autophagy-related proteins (LC-3 and p62) in granulation tissue showed that the values of LC3 and LC3-II/LC3-I and the expression of LC3 gene in wound granulation tissue of PRP group were significantly higher than those before treatment (P < .05). The value and gene expression of P62 protein were lower than those before treatment (P < .05). In the control group, there was no significant difference in LC3 and P62 protein gray level and gene expression before and after treatment (P > .05). The level of autophagy in the wound of PRP group increased after treatment, while there was no statistical significance in the control group. The results of ELISA showed that the concentration of IL-6 in granulation tissue of the PRP treatment group was lower than that before treatment (P < .05), while there was no significant difference in IL-6 in the control group after treatment. The concentration of IL-10 increased in both groups after treatment, but the concentration in PRP group was higher than that in control group (P < .05). Conclusions: This study shows that PRP gel has advantages in accelerating wound healing, relieving pain, shortening healing time and reducing inflammatory response in treating diabetic foot ulcers wound, which may be related to autophagy, and provides new ideas for the treatment of diabetic foot ulcers.

Keywords

PRP diabetic foot ulcers < lower extremity wound platelet-rich plasma autophagy

Introduction

The incidence of diabetes mellitus in the world is currently increasing year by year. Data from the sixth edition of the diabetes map published by the World Diabetes Federation (IFD) in 2013 showed that there were 382 million people with diabetes worldwide in 2013. Diabetic foot is a serious complication of diabetes mellitus, which is one of the important causes of disability and death. About 12% to 25% of diabetic patients will suffer from diabetic foot disease in their lifetime. Its long treatment time, high cost and heavy burden on patients and their families have become one of the urgent public health problems in the world.¹

The pathogenesis of diabetic foot is mainly caused by multiple factors such as blood vessels, nerves, infection and so on, which lead to the change of local micro-environment, so that the normal wound healing period is prolonged or even does not heal. A number of studies have shown that the reduction of local secretion of diabetic foot substances such as platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor (TGF) and so on is an important reason for the chronic refractory wound.²

Autophagy was first proposed in 1966, is a process that degrades and recycles damaged proteins, organelles and pathogens, thus playing an important role in maintaining cell differentiation, environmental homeostasis and immune regulation etc. Autophagy is associated with many systemic diseases,^3–5 and its influence on the quality, structure, quantity and insulin secretion of insulin β-cells can accelerate the progress of diabetes-related complications.⁶ Wound healing is a complex, multistep biological process involving multiple cell types, which can be complicated by the hyperglycemic and hypoxic environment of diabetic wounds. Autophagy plays an important role in wound healing by promoting the activation of inflammatory cells, enhancing their anti-inflammatory and anti-infection activities, and promoting angiogenesis.⁷ In the inflammatory phase, autophagy has an anti-infective effect, and it will negatively regulate the inflammatory response, thereby preventing excessive inflammation that will lead to tissue damage.⁸ In the proliferative stage, local hypoxia can induce autophagy in the wound, and play a role in anti-apoptosis and anti-oxidative stress to promote cell survival.⁹ Autophagy of vascular endothelial cells promotes wound angiogenesis and is conducive to the completion of wound re-epithelialization.¹⁰ Limited studies have shown that the induction of autophagy has a certain relationship with the healing of diabetic wounds.^11,12 The regulation of autophagy may be one of the new strategies for the treatment of diabetic foot.

Platelet-rich plasma (PRP), as the first generation of platelet concentration preparation, has been widely used in plastic surgery, oral and maxillofacial surgery, rehabilitation department and other fields. With the activation of platelet α-granule, a large number of active substances such as VEGF, PDGF and TGF are released to promote wound healing and have a good effect in diabetic foot.¹³ In vitro model of diabetic infection, PRP also promotes the release of anti-inflammatory cytokines and inhibits the activation of inflammatory cytokines to achieve antibacterial effect,¹⁴ but its specific physiological mechanism has not been thoroughly studied. In the field of rehabilitation, PRP can induce autophagy and promote cartilage protection, but some scholars have put forward different opinions.^15,16 Some studies have shown that PRP can regulate autophagy through Mir-223 /PAQR3 signaling pathway, but the specific activation mechanism still needs to be further studied.¹⁷

In this study, we designed clinical trials to observe the clinical effect of PRP gel in treating diabetic foot ulcers from the aspects of wound healing rate, healing time, pain, etc, and to detect the expression of autophagy and inflammation, to explore the potential relationship between PRP in the treatment of diabetic foot ulcers and autophagy, to improve the theoretical basis of PRP for diabetic foot ulcers, and to provide a new target for the treatment of diabetic foot.

Method

the Research Object

From 2019 to 2020, We conducted a prospective study. We selected 30 patients with diabetic foot who were hospitalized in the Plastic Surgery Department, and randomly divided them into 2 groups. The method of random grouping is computer random number method. All participating patients agreed to PRP or conventional treatment. There were 15 patients in PRP treatment group and 15 patients in control group. Inclusion criteria were18 to 65 years old patients with diabetic foot regardless of gender who agreed to PRP treatment, diabetic foot wagner grade II to IV, the wound area greater than 2 cm², ankle-brachial index greater than 0.9. Wagner grade I, There were some ulcers on the foot, but there was no infection. Wagner grade II, There was a co-infected ulcer. Wagner grade III, A deep abscess developed, along with severe soft tissue, fascia, and bone infection. Wagner grade IV, There are local manifestations of gangrene. Wagner grade V, The whole foot gangrene.¹⁸ Exclusion criteria were patients with combined cardiovascular and cerebrovascular diseases; Patients with combined malignant tumors; patients using steroids, chemotherapy or immunosuppressant; Patients with liver and kidney failure; Patients with lower limb vascular disease and other infectious diseases. There were 16 males and 14 females with an average age of 53 years old. The average initial area of diabetic foot ulcer was 11.07 cm², of which the minimum was 3.8 cm² and the maximum was 16.7 cm². This study was reviewed by the Ethics committee of the institution.

PRP Preparation

The preparation method is a commercial preparation method, using a platelet-rich plasma preparation set (Weigao, Shandong, Registration Certificate Number: China Food and Drug Administraion (2013) No.3410051) for PRP preparation. Preparation process: Step 1, 30–40 ml of the patient's venous blood was collected with the collection vessel set; Step 2, place in a centrifuge and centrifuged by secondary centrifugation. Step 3, after the upper PPP (platelet-poor plasma) was extracted, the liquid PRP and thrombin (1000u:1 ml) were simultaneously and uniformly dropped into the patient's wound surface 1:1 ratio to make PRP gel.

Treatment Methods

After hospitalization, all patients with diabetic foot received standard blood glucose control. Debridement was performed before treatment to remove surface necrotic tissue and rinsed with saline. All patients were hospitalized and bedridden until healing was achieved. PRP treatment group: after cleaning the wound surface, the prepared PRP gel was evenly covered to the DF wound and fixed with Vaseline gouze. Removing the dressing and cleaning the wound after 2 days, then routinely dressing change once in 1 to 2 days, and repeat the PRP treatment at day 7. Patients in the PRP group received a total of two PRP treatments. The control group also thoroughly cleaned the wound, covered with saline sterile gauze, and bandaged with Vaseline gauze. Also, the gauze debridement was opened after 2 days, and the medicine was also changed at the frequency of 1 to 2 days / time.Skin grafting was performed when patients in both groups reached suitable conditions.

Clinical Data Collection

1. General data collection: the medical history of patients in the two groups was inquired and specialized physical examination was performed. All patients’ names, gender, age, BMI, blood glucose value, glycosylated hemoglobin, DF ulcer area, DF grade, and the course of diabetes were recorded. It was done by the same physician. 2. Wound healing rate:(1) A Canon 6D camera is used to capture images perpendicular to the DF wound surface with a fixed high variation of 15 cm. (2) The size of the wound was analyzed by the image software Image J. (3) Images were collected before treatment, and 7, 14 and 21 days after treatment. (4) Calculation formula of wound healing rate: initial area minus post-treatment area divided by initial treatment area. 3. Pain indicators: the VAS pain score table was used. (1) Mild pain: 3 points below. (2) Moderate pain, which interferes with sleep hut tolerable: 4–6 points. (3) Severe pain, which affects appetite and sleep is unbearable: 7–10 points. (4) The patient's pain was collected for 5 consecutive days. 4. Wounding healing time: the time from admission to reaching the cure standard was recorded for the two groups of patients. The cure standard was: (1) Patients’ blood glucose was well controlled. (2) After skin grafting, the wound skin was closely adhered to and survived well with no deep tissue exposure and no exudation of secretions. 5. Autophagy protein detection:(1) Tissue collection method: After the wound secretion was removed, a small amount of granulation tissue was taken from the DF wound and stored at −80 °C. (2) Proteins detected: microtubule-associated protein light chain 3(LC-3) and ubiquitin-binding protein (p62), which is related to autophagy. (3) Laboratory method: Western blot(WB) and PCR are used to quantitatively determine the expression levels of autophagy-related proteins in granulation tissues. 6. Determination of inflammatory factors: the expression level of inflammatory factors was determined by enzyme-linked immunosorption assay.

Statistical Method

SPSS Statistics 23.0 is used for processing, and the measurement data are described by (x ± s). The t-test is used for the measurement data between the two samples, and the chi-square test was used for the enumeration data. The test level α = 0.05, and P < .05 was considered statistically significant.

PCR Primers Used in the Study

Primer	Sequences (5′→3′)
LC3	F-AATCCCGGTGATCATCGAGC R-GCCGGATGATCTTGACCAAC
P62	F-TCGGATAACTGTTCAGGAGGAG R-TCGGATTCTGGCATCTGTAGG
GAPDH	F-AACGGATTTGGTCGTATTGGG R-TCGCTCCTGGAAGATGGTGAT

Results

General Data

The subjects were 30 patients hospitalized in plastic surgery department during September 2018 and 2020, randomly divided into two groups, 15 in the PRP treatment group and 15 in the control group. There were 16 males and 14 females with an average age of 53, a maximum age of 64 and a minimum age of 40. The initial area of diabetic foot ulcers averaged 11.07 cm², with the smallest 3.8 cm² and the largest 16.7 cm². In terms of general information, there were no statistical differences in gender composition, age, body mass index (BMI), diabetes course, initial DF ulcer area, and glycation hemoglobin (HbAlc) (P > .05) as detailed in Table 1.

Table 1.

Comparison of General Data Between Prp Group and Control Group.

Parameter	PRP group (n = 15)	Control group (n = 15)	Statistic	P value
Gender: male/female	9/6	7/8	χ2 = 0.536	P = .464
Age(years)	51.93 ± 8.439	54.07 ± 7.363	t = 0.738	P = .467
BMI (kg/m²)	24.50 ± 2.01	23.69 ± 1.82	t = 1.145	P = .262
Diabetes course (year)	6.06 ± 1.50	5.50 ± 1.17	t = 1.123	P = .271
Initial DF ulcer area (cm²)	10.09 ± 2.70	12.07 ± 3.71	t = 1.172	P = .11
HbAlc (%)	8.18 ± 0.44	8.06 ± 0.48	t = 1.18	P = .47

Wound Healing Rate

In the PRP group, three patients were eligible for skin grafting 21 days earlier and underwent skin grafting, and the fourth image was taken before grafting. After treatment of DF wounds in both groups, the oozing of necrosis tissue and secretions decreased, the area of ulcers decreased, and the overall healing trend was shown in Table 2 and Figure 1. Statistical difference between the two treatments occurs on the seventh day (P < .05), and the healing rate in the PRP treatment group was higher on the seventh, fourteenth and twenty-first days than that in the control group (t = 5.246, t = 12.772, t = 35.056). On the twenty-first day, compares with the control group (0.204 ± 0.013), the wound healing rate of PRP treatment group (0.396 ± 0.014) was more effective in promoting wound healing. (Figure 2)

Table 2.

Wound Healing Rate of PRP Group and Control Group.

Time	PRP group	Control group	Statistic	P
day 7	0.204 ± 0.054	0.122 ± 0.027	t = 5.246	P < .05
day14	0.314 ± 0.038	0.164 ± 0.018	t = 12.772	P < .05
day21	0.396 ± 0.014	0.204 ± 0.013	t = 35.056	P < .05

Pain Indicator

VAS scores for both groups generally showed a downward trend and there was no significant difference between the first and second day (P > .05). At the beginning of the third day, patients treated with PRP scored lower on VAS pain values and pronounced more pain relief than those in the control group. On the fifth day after treatment, the PRP group VAS pain score (2.90 ± 0.39) was significantly lower than that of the control group (4.70 ± 0.31) and significantly improved the pain compared to the first day (5.90 ± 0.31). Figure 3 for details.

Time of Healing

The healing time of PRP group and control group was (31.40 ± 4.47) days and (43.20 ± 5.03) days, respectively, there was a statistical difference between those groups (P < .05), and the healing time of PRP treated patients was shorter.

Autophagy Expressions

Western Blot

LC3

The values of the two groups of LC3-II/GAPDH (0.3692 ± 0.0471, 0.3942 ± 0.0451) were not statistically significant prior to treatment (P > .05). In the PRP group, there was a significant difference in the values of LC3-II/GAPDH before and after treatment (0.3692 ± 0.0471, 0.6805 ± 0.0737) (P < .05). Further analysis of the LC3-II/LC3-I ratio found that in the PRP group, the value of LC3-II/LC3-I after treatment (1.6984 ± 0.1283) was higher than before treatment (0.6913 ± 0.1133) (P < .05). There was no significant difference in the values of LC3-II/GADPH and LC3-II/LC3-I before and after treatment in the control group (P > .05). Both LC3-II/GAPDH and LC3-II/LC3-I in PRP group had higher values than that in the control group (P < .05) after treatment. The results above showed that there is an increase in autophagy levels after PRP treatment and no significant change in autophagy levels after treatment in the control group. Figures 4 to 6, Tables 3 and 4.

Table 3.

LC3-II/GAPDH Grey Value in PRP Group and Control Group.

	Before treatment	After treatment
PRP group (n = 15)	0.3692 ± 0.0471	0.6805 ± 0.0737
Control group (n = 15)	0.3942 ± 0.0451	0.4177 ± 0.0402

Table 4.

LC3-II/LC3-I Grey Value in PRP Group and Control Group.

	Before treatment	After treatment
PRP group (n = 15)	0.6913 ± 0.1133	1.6984 ± 0.1283
Control group (n = 15)	0.7107 ± 0.0778	0.6935 ± 0.571

P62

The values of the P62 protein before and after treatment are shown in Figure 7 and 8, Table 5. The values of the two groups of P62/GAPDH were not statistically significant (P > .05) prior to treatment (0.7361 ± 0.0556, 0.7127 ± 0.0323). The analysis found that the value of PRP group was significantly lower after treatment (0.4431 ± 0.0396) than before (0.7361 ± 0.0556), and was statistically significant (P < .05). Meanwhile, there was no significant change in the control group (P > .05). The value of P62/GAPDH in PRP group after treatment was lower than that in the control group (P < .05). The results above further showed that the autophagy level of the wound granulation tissues increased after PRP treatment, while the autophagy level of the control group did not change significantly.

Table 5.

P62/GAPDH I Grey Value in PRP Group and Control Group.

	Before treatment	After treatment
PRP group (n = 15)	0.7361 ± 0.0556	0.4431 ± 0.0396
Control group (n = 15)	0.7127 ± 0.0323	0.7296 ± 0.3183

PCR

Using PCR to detect the expression of LC3 and P62 genes in each group, Statistically, the expression of LC3 genes increased after treatment in the PRP treatment group (P < .05), There was no significant change in the control group (P > .05). The amount of expression after gene therapy for P62 decreased in the PRP treatment group (P < .05).

The expression of P62 after treatment decreased in the PRP group (P < .05), Tables 6 and 7, Figure 9 and 10 for details.

Table 6.

LC3 Expression in PRP Group and Control Group by PCR.

	Before treatment	After treatment
PRP group (n = 15)	0.7075 ± 0.1900	1.16195 ± 0.2488
Control group (n = 15)	0.7035 ± 0.1846	0.7131 ± 0.1791

Table 7.

P62 Expression in PRP Group and Control Group by PCR.

	Before treatment	After treatment
PRP group (n = 15)	1.2791 ± 0.2395	1.0947 ± 0.1699
Control group (n = 15)	1.2901 ± 0.2456	1.2717 ± 0.2262

Inflammatory Factors

IL-6

There was no statistical difference in the concentration of IL-6 in the PRP treatment group and the control group prior to treatment (P > .05). The concentration of IL-6 achieves (0.9174 ± 0.1296) pg/ml in the PRP group in comparison to (1.2754 ± 0.1774) pg/ml in the control group after treatment, and there were statistical differences between the two groups (P < .05), as detailed in Table 8. The concentration of IL-6 in the PRP group was significantly lower than that in the control group and showed a downward trend compared to the pre-treatment.

Table 8.

IL-6 in PRP Group and Control Group by Elisa.

	Before treatment (pg/ml)	After treatment(pg/ml)	Statistic	P value
PRP group（n = 15）	1.3118 ± 0.1817	0.9174 ± 0.1296	t = 6.846	P < .05
Control group(n = 15)	1.2971 ± 0.1521	1.2754 ± 0.1774	t = 0.360	P = .721
P value	P = .812	P < .05

IL-10

The concentration of IL-10 in the PRP group was (1.5801 ± 0.072) pg/ml and (1.5044 ± 0.4429) pg/ml in the control group after treatment, with statistical differences between the two groups (P < .05), as detailed in Table 9. After treatment in both groups, the concentration of IL-10 in the tissue of the sprouts increased compared to the pre-treatment level (P < .05). After treatment, the concentration of IL-10 in the PRP treatment group was higher than that in the control group (P < .05).

Table 9.

IL-10 in PRP Group and Control Group by Elisa.

	Before treatment (pg/ml)	after treatment(pg/ml)	Statistic	P value
PRP group(n = 15)	1.3879 ± 0.0607	1.5801 ± 0.072	t = 7.899	P < .05
Control group(n = 15)	1.4047 ± 0.0387	1.5044 ± 0.4429	t = 6.563	P < .05
P	P = .375	P < .05

Discussion

Autologous platelet-rich plasma is the first generation of platelet concentrate, which has been widely used in plastic surgery, oral and maxillofacial surgery and rehabilitation department, and also plays an important role in the treatment of diabetic foot wounds.

The Meta-analysis of 525 participants (270 of whom were treated with PRP) by Tasmania del Pino-Sedeño on epithelial- forming area, ulcer volume, wound healing time and complication rate showed that PRP was more effective and safer in the treatment of diabetic foot than conventional antibacterial dressings.¹⁹ Common methods of PRP treatment are local injections and gel coverage, local injections is injecting liquid preparation into wound substrate and invasive dermal tissue, to activate the activation of cells in the peripheral tissue and promote wound healing, but this kind of method is painful for diabetic foot patients without incorporating sensory nerve abnormalities. This study adopted the method of PRP gel coverage, which can relieve the pain of patients, and can make the reagent adhere to the wound more closely and release various growth factors more permanently than the injection method. Previous studies have also shown that gelatinous PRP has superior antibacterial and healing effects.

In this study, patients who treated with PRP gel were different in the pain experience from day 3 compared with the control group, and the trend of further mitigation appears. In the comparison of wound healing rate, there was a statistical difference between the PRP group and the control group on the seventh day (P < .05) and the results on the 14th and 21st day were significantly higher than the control group. This is consistent with the results that we tracked the healing time of patients in the two groups, namely the PRP group (31.40 ± 4.47) days and the control group (43.20 ± 5.03)days.

PRP has the advantage of reducing pain, accelerating wound healing, shortening healing time and making it easy to operate in the clinical treatment of diabetic foot. However, there is no unified standard for the preparation of PRP at present, the common centrifugal methods include primary and secondary centrifugal methods. The efficacy of PRP prepared with different centrifugal parameters also needs to be studied further. At the same time, selection of secondary dressing after PRP treatment is also critical. Only Naoki Morimoto et al are currently conducting studies on the effects of secondary dressings on PRP efficacy and the final results are not yet available.²⁰ In the selection of secondary wound dressing, the principle of good fixation of PRP preparation and avoiding changes in the biological activity of PRP preparation should he followed, and further studies are necessary in the future.

wound healing is regulated by various growth factors and cytokines secreted by platelets and white blood cells in fibrin.²¹ Due to a variety of internal and external factors, which leads to the accumulation of wound inflammatory factors, lack of oxygen, nutrient deficiency, infection of Diabetic foot patients, the normal healing process was destroyed and gradually forming a chronic difficult healing wound surface.²² The release and activation of various growth factors is the main mechanism by which PRP preparations promote DFU wound healing.²³ In recent years, the physiological mechanism of PRP has been further supplemented. Some studies have shown that the antibacterial properties of PRP also play an important role in the healing, but whether the white blood cells it contains play an antibacterial effect are still in dispute.^24,25 Accumulation of inflammation factor is one of the important factors to aggravate DF wound tissue damage,²⁶ by establishing a wound model of in vitro diabetes infection, Li Tao et al further confirmed that PRP has the synergy effects in inhibiting inflammatory factors and promoting anti-inflammatory factors,¹⁴ there are other studies show that PRP has the effect of improving local inflammatory response, but he specific pathway of PRP control of inflammation are still unclear.

The classical autophagy process starts with stimulation from oxidative stress, infection, lack of nutrients and growth factors, forms biomembrane vesicles in the cytoplasm, then binds to lyophage into autophagy lysosomes, and eventually its rich proteollase degrade the contents of the autophagage. The exact role and regulation of autophagage in the healing process of diabetic foot ulcer are still controversial. In this study, the expression levels of LC3 (microtubule-associated protein light chain3) and P62 (ubiquitin-binding protein) before and after the treatment of diabetic foot ulcer by PRP were measured to explore the influence of PRP on autophagy. When autophagy is activated, LC3 protein in cytoplasm is partially enzymolvzed by Agt-7 to produce LC3-I and then reacts with phosphatidy ethanolamines to produce LC3-II, which binds to the autophagy microextracorporeal membrane. This is a necessary process for the formation of autophagosomes and the selection of substrates for degradation.²⁷ Therefore, the expression level of LC3-II and the ratio of LC3-II to LC3-I are often used to reflect the strength of autophagy. P62 protein is an autophagy receptor with an ubiquitin-binding domain, which binds to autophagy substrate and LC3-II on the lysosome and promotes the expansion of phagocytes around the autophagy substrate, and ultimately degrades the contents. Therefore, whether the degradation of P62 can also reflect the occurrence of autophagy.²⁸

Our study found that the level of autophagy increased in diabetic foot wounds after PRP treatment, while there was no statistically significant change in the control group. In this experiment, although there was no statistical change in autophagy level in the control group after treatment, both P62 and LC3-II/LC3-1 showed a trend of decreasing autophagy level. Therefore, we speculate that the decreased autophagy level is one of the factors delaying the wound healing of diabetic foot ulcer but a larger sample size is needed for further study. The clinical indicators of this study showed that wound healing was faster after PRP treatment, thus revealing a new physiological mechanism for PRP treatment of diabetic foot. In conclusion, PRP may accelerate the healing of diabetic foot ulcer wounds by increasing levels of autophagy.

Inflammation response is also critical in the progression of diabetic foot ulcers. The normal wound healing process of diabetic patients is disrupted, and the accumulation of a large number of inflammatory factors such as IL-6, TNF-α, NO, IL-1 makes it become chronic and refractory wound and aggravates tissue damage.²⁶ As an anti-inflammatory factor, IL-10 can promote wound healing by inhibiting inflammatory response.²⁹ In this experiment, we found that for patients with diabetic foot treated by PRP, anti-inflammatory factor IL-6 was decreased after treatment (0.9174 ± 0.1296 pg/ml) compared with pre-treatment (1.3118 ± 0.1817 pg/ml) (P < .05),but had no significant change in the control group(P > .05). The anti-inflammatory factor IL-10 showed an increasing trend after treatment (1.5801 ± 0.072 pg/ml) (P < .05). In the control group, IL-10 in diabetic foot patients after routine dressing change showed the same increasing trend (P < .05), but the concentration of IL-10 in PRP treatment group (1.5801 ± 0.072) was higher than that in the control group (1.5044 ± 0.4429)(P < .05). Present studies have reported that PRP can promote anti-inflammatory factors and inhibit pro-inflammatory factors in vitromodels,¹⁴ but the specific mechanism remains unclear. As a homeostasis mechanism, autophagy plays a negative role in the regulation of inflammatory response by affecting the development, homeostasis and survival of inflammatory cells.³⁰ As an important anti-inflammatory factor, IL-10 has been found that autophagy can promote its expression, but the relationship between them is affected by the environment.³¹ In this experiment, we found that the autophagy level and the anti-inflammatory factor IL-10 in the DF wound tissue of patients in the PRP group were simultaneously increased after treatment, accompanied by the decrease of the inflammatory factor IL-6. Therefore, we speculate that PRP can improve the micro-environment of diabetic foot wounds and accelerate the healing by inhibiting the inflammatory response. This process may be related to autophagy, which still needs further study in the future.

Conclusion

This study demonstrated that platelet-rich plasma(PRP) has a good effect in the treatment of diabetic foot ulcer without adverse reactions. It has the advantages of simple operation, relieving pain of patients, accelerating wound healing and shortening healing time. We speculated that PRP might improve the local micro-environment and accelerate wound healing by increasing the level of autophagy and regulating the inflammatory response of diabetic foot wounds. This study is limited by the sample size, and multi-center trials can be carried out in the future to explore the relationship between PRP and autophagy. At the same time, animal models can be constructed to clarify the specific role of autophagy in each stage of diabetic foot wound healing, and autophagy drugs can be developed to treat diabetic foot ulcers by targeted regulation of autophagy.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Pengyu Zhao

References

Wang

, et al. Prevalence and control of diabetes in Chinese adults[J]. JAMA. 2013;310(9):948‐959.

Musuuza

Sutherland

Kurter

, et al. A systematic review of multidisciplinary teams to reduce major amputations for patients with diabetic foot ulcers[J]. J Vasc Surg. 2020;71(4):1433‐1446.e3.

Cosialls

Mehrpour

, et al. Crosstalk between autophagy and metabolic regulation of cancer stem cells[J]. Mol Cancer. 2020;19(1):27.

Fan

Zhao

, et al. LncRNA BDNF-AS promotes autophagy and apoptosis in MPTP-induced Parkinson’s disease via ablating microRNA-125b-5p[J]. Brain Res Bull. 2020 Apr;157:119‐127. doi:10.1016/j.brainresbull.2020.02.003.

Dong

Chen

Gao

, et al. Molecular machinery and interplay of apoptosis and autophagy in coronary heart disease[J]. J Mol Cell Cardiol. 2019 Nov;136:27‐41. doi:10.1016/j.yjmcc.2019.09.001.

Bugliani

Mossuto

Grano

, et al. Modulation of autophagy influences the function and survival of human pancreatic beta cells under endoplasmic Reticulum stress conditions and in type 2 diabetes[J]. Front Endocrinol (Lausanne). 2019 Feb 26;10:52. doi:10.3389/fendo.2019.00052.

Ren

, Zhao F, Zhang Q, et al. Autophagy and skin wound healing. Burns Trauma. 2022 Feb 16;10:tkac003. doi:10.1093/burnst/tkac003.

Park

Shrestha

Youn

, et al. Autophagy primes neutrophils for neutrophil extracellulartrap formation during sepsis. Am J Respir Crit Care Med. 2017 Sep 1;196(5):577-589. doi:10.1164/rccm.201603-0596OC.

Zhang

Jiang

, et al. Involvement of autophagy in hypoxia-BNIP3 signaling to promote epidermal keratinocyte migration. Cell Death Dis. 2019 Mar 8;10(3):234. doi:10.1038/s41419-019-1473-9.

10.

, Liu WJ, Xue P, et al. Autophagy promotes MSC-mediated vascularization in cutaneous wound healing via regulation of VEGF secretion. Cell Death Dis. 2018;9(2):58.

11.

Han

Sun

Han

, et al. Clinical perspectives on mesenchymal stem cells promoting wound healing in diabetes mellitus patients by inducing autophagy[J]. Eur Rev Med Pharmacol Sci. 2015;19(14):2666‐2670.

12.

Jin

Zhang

Wang

, et al. Melatonin protects endothelial progenitor cells against AGE-induced apoptosis via autophagy flux stimulation and promotes wound healing in diabetic mice[J]. Exp Mol Med. 2018;50(11):154.

13.

Del

Trujillo

Andia

, et al. Platelet-rich plasma for the treatment of diabetic foot ulcers: A meta-analysis[J]. Wound Repair Regen. 2019;27(2):170‐182.

14.

Wang

, et al. Platelet-rich plasma plays an antibacterial, anti-inflammatory and cell proliferation-promoting role in an in vitro model for diabetic infected wounds[J]. Infect Drug Resist. 2019 Jan 29;12:297-309. doi:10.2147/IDR.S186651.

15.

Moussa

Lajeunesse

Hilal

, et al. Platelet rich plasma (PRP) induces chondroprotection via increasing autophagy, anti-inflammatory markers, and decreasing apoptosis in human osteoarthritic cartilage[J]. Exp Cell Res. 2017;352(1):146‐156.

16.

Yang

Yin

, et al. Autophagy is independent of the chondroprotection induced by platelet-rich plasma releasate[J]. Biomed Res Int. 2018 Jul 24;2018:9726703. doi:10.1155/2018/9726703.

17.

Yang

Peng

. Platelet rich plasma alleviates OGD/R injury in N2a cell by enhancing autophagy through the miR223/PAQR3 pathway. Acta Neurobiol Exp (Wars). 2022;82(2):121‐132.

18.

Shah

, Inturi R, Anne D, et al. Wagner’s classification as a tool for treating diabetic foot ulcers: Our observations at a suburban teaching hospital. Cureus. 2022;14(1):e21501.

19.

Del

Tru

Andia

, et al. Platelet-rich plasma for the treatment of diabetic foot ulcers: A meta-analysis[J]. Wound Repair Regen. 2019;27(2):170‐182.

20.

Morimoto

Kakudo

Matsui

, et al. Exploratory clinical trial of combination wound therapy with a gelatin sheet and platelet-rich plasma in patients with chronic skin ulcers: Study protocol[J]. BMJ Open. 2015;5(5):e007733.

21.

Janis

Harrison

. Wound healing: Part I. Basic science. Plast Reconstr Surg [J]. 2016;138(3 Suppl):9S‐17S.

22.

Aumiller

Dollahite

. Pathogenesis and management of diabetic foot ulcers[J]. JAAPA. 2015;28(5):28‐34.

23.

Liao

Marra

Rubin

. Application of platelet-rich plasma and platelet-rich fibrin in fat grafting: Basic science and literature review[J]. Tissue Eng Part B Rev. 2014;20(4):267‐276.

24.

Cieslik-Bielecka

Bold

Ziółkowski

, et al. Antibacterial activity of leukocyte- and platelet-rich plasma: An in vitro study[J]. Biomed Res Int. 2018 Jun 27;2018:9471723. doi:10.1155/2018/9471723.

25.

Cetinkaya

Yenilmez

Petrone

, et al. Platelet-rich plasma as an additional therapeutic option for infected wounds with multi-drug resistant bacteria: In vitro antibacterial activity study[J]. Eur J Trauma Emerg Surg. 2019;45(3):555‐565.

26.

Dhamodharan

, Viswanathan V, Krishnamoorthy E, et al. Genetic association of IL-6, TNF-alpha and SDF-1 polymorphisms with serum cytokine levels in diabetic foot ulcer. Gene. 2015;565(1):62‐67.

27.

Jiang

Zou

Zhu

, et al. SIP/CacyBP promotes autophagy by regulating levels of BRUCE/apollon, which stimulates LC3-I degradation[J]. Proc Natl Acad Sci U S A. 2019;116(27):13404‐13413.

28.

Yim

Mizushima

. Lysosome biology in autophagy[J]. Cell Discov. 2020 Feb 11;6:6. doi:10.1038/s41421-020-0141-7.

29.

Saraiva

Vieira

O’Garra

. Biology and therapeutic potential of interleukin-10[J]. J Exp Med. 2020;217(1):e20190418.

30.

Qian

Fang

Wang

. Autophagy and inflammation[J]. Clin Transl Med. 2017;6(1):24.

31.

Yao

. Interactions between autophagy and inhibitory cytokines[J]. Int J Biol Sci. 2016;12(7):884‐897.