Efficacy of Huangma Ding or autologous platelet-rich gel for the diabetic lower extremity arterial disease patients with foot ulcers

Abstract

BACKGROUND

Diabetes foot is one of the most serious complications of diabetes and an important cause of death and disability, traditional treatment has poor efficacy and there is an urgent need to develop a practical treatment method.

AIM

To investigate whether Huangma Ding or autologous platelet-rich gel (APG) treatment would benefit diabetic lower extremity arterial disease (LEAD) patients with foot ulcers.

METHODS

A total of 155 diabetic LEAD patients with foot ulcers were enrolled and divided into three groups: Group A (62 patients; basal treatment), Group B (38 patients; basal treatment and APG), and Group C (55 patients; basal treatment and Huangma Ding). All patients underwent routine follow-up visits for six months. After follow-up, we calculated the changes in all variables from baseline and determined the differences between groups and the relationships between parameters.

RESULTS

The infection status of the three groups before treatment was the same. Procalcitonin (PCT) improved after APG and Huangma Ding treatment more than after traditional treatment and was significantly greater in Group C than in Group B. Logistic regression analysis revealed that PCT was positively correlated with total amputation, primary amputation, and minor amputation rates. The ankle-brachial pressure and the transcutaneous oxygen pressure in Groups B and C were greater than those in Group A. The major amputation rate, minor amputation rate, and total amputation times in Groups B and C were lower than those in Group A.

CONCLUSION

Our research indicated that diabetic foot ulcers (DFUs) lead to major amputation, minor amputation, and total amputation through local infection and poor microcirculation and macrocirculation. Huangma Ding and APG were effective attreating DFUs. The clinical efficacy of Huangma Ding was better than that of autologous platelet gel, which may be related to the better control of local infection by Huangma Ding. This finding suggested that in patients with DFUs combined with coinfection, controlling infection is as important as improving circulation.

Key Words: Diabetic foot ulcer, Huangma Ding, Autologous platelet-rich gel, Ankle-brachial index, Transcutaneous oxygen partial pressure

Core Tip: We investigated whether Huangma Ding or autologous platelet-rich gel (APG) treatment would benefit diabetic lower extremity arterial disease patients with foot ulcers. Our research indicated that diabetes foot ulcers lead to major amputation, minor amputation, and total amputation through local infection and poor microcirculation and macrocirculation. Huangma Ding and APG were effective at treating diabetic foot ulcers. The clinical efficacy of Huangma Ding was better than that of autologous platelet gel, which may be related to the better control of local infection by Huangma Ding.

INTRODUCTION

In recent times, there has been a rise in diabetes incidents globally. As of 2021, according to the International Diabetes Federation, the global diabetic population was estimated at 537 million, marking a 1.2% growth over the preceding five years. One of the critical complications arising from diabetes is diabetic foot ulcers (DFUs). These ulcers are believed to have an occurrence rate of 6.3%, impacting between 9.1 and 26.1 million individuals annually[1]. Diabetes can cause peripheral neuropathy and vascular damage, resulting in conditions such as foot ulcers and Charcot joint disease. These complications are susceptible to secondary infections, which can necessitate amputation (either minor or major). Roughly 10% of individuals with these conditions pass away within a year of being diagnosed.

In patients suffering from DFUs, wound infection stands as a significant indicator for the likelihood of amputation in the lower extremities[2]. The majority of wound infections necessitate surgical debridement, and between 15% to 20% of these instances could result in amputation. For patients experiencing severe infections or osteomyelitis, the likelihood of amputation surges to around 90%[3]. Dysfunction in the immunological and inflammatory responses among patients with DFUs is a primary factor contributing to the ineffective treatment outcomes of DFU.

In China, individuals with diabetic foot conditions experience a significant incidence of lower extremity vascular lesions. While interventions targeting these vascular issues show some initial effectiveness, the high rate of restenosis and the complexity of these procedures result in a considerable number of diabetic patients being readmitted and undergoing reamputation. Our prior cohort study highlighted that although interventional techniques in DFUs with lower extremity ischemia can decrease the rate of major amputations, they have a minimal impact on the more frequently occurring minor amputations in clinical settings. Autologous platelet-rich gel (APG) enhances microcirculation, thereby more effectively promoting wound healing and reducing minor amputation rates in patients with DFUs who also suffer from lower extremity ischemia. However, the preparation of APGs is complex and not readily feasible for clinical use in primary care hospitals.

Within the framework of traditional Chinese medicine (TCM), DFUs are categorized under the conditions of arthralgia syndrome and gangrene. Typically, these patients exhibit a deficiency in qi, which contributes to a deficiency in Xue (blood), resulting in weakened Yang energy. This imbalance causes a loss of warmth and harmony, leading to cold coagulation, and stagnation of blood and vessels, which ultimately may result in limb necrosis over time. In clinical settings, TCM is increasingly recognized as a supportive treatment option for managing DFU[4,5]. Huangma Ding is a blend of Nux vomica and Coptidium, as per the principles of traditional Chinese medicine. Nux vomica is known for its bitter and cold properties, offering benefits such as anti-infection capabilities, nodule dispersion, blood and arterial circulation maintenance, and pain relief. Similarly, species of Coptidium, with their bitter and cold qualities, are believed to clear heat and eliminate fire, remove dampness, boost Yang and Qi, enhance immune function, combat infections, and support the circulation of blood and the health of arteries[6,7]. Huangma Ding originates from the traditional recipe “Huangma liquor” and is a concoction prepared internally by the Chongqing Traditional Chinese Medicine Hospital[8]. Extensively employed in wound healing, Huangma Ding is believed to possess anti-infective properties and facilitate the healing process of local wounds. This study aims to evaluate the therapeutic effectiveness and rate of amputation between Huangma Ding^® and APG in individuals with DFUs, while also investigating the potential underlying mechanisms involved.

MATERIALS AND METHODS

Research subjects and grouping

The research protocol received approval from the Ethics Committee of Dazu Hospital, Chongqing Medical University, and The People’s Hospital of Dazu, adhering to the principles outlined in the Declaration of Helsinki. Prior to participation, all patients provided informed consent by signing the respective consent forms.

The research subjects for this study consisted of patients with diabetic foot tissue who were admitted to various hospitals, including Dazu Hospital Affiliated with Chongqing Medical University, First Affiliated Hospital of Army Medical University, Cancer Hospital Affiliated with Chongqing University, and People’s Hospital of Yubei District, during the period from July 2017 to October 2022. The patient inclusion criteria were as follows: (1) Individuals diagnosed with either type 1 or type 2 diabetes; (2) age range of 18-75 years; (3) presence of diabetic foot categorized under the Wagner I-V grade; (4) willingness and ability to provide informed consent; and (5) ankle-brachial pressure (ABI) within the range of 0.6-1.2. On the other hand, the patient exclusion criteria were as follows: (1) Non-diabetic individuals; (2) pregnant or lactating patients; (3) severe functional failure of major organs; (4) presence of severe life-threatening diseases; and (5) mental illness that would hinder participation in treatment and follow-up.

The decision regarding amputation was made by the orthopedic surgeon considering the patient’s preference for immediate treatment. Prior to study enrollment, each patient was assessed by their physician based on these criteria.

A total of 155 diabetic foot patients with foot ulcers were enrolled in the study. These patients met the 1999 WHO diagnostic criteria for DFU, as determined through a comprehensive assessment including medical history, physical examination, medication, and hormone history, as well as blood test results. Physicians diagnosed all patients and recommended the addition of Huangma Ding or APG as part of their essential treatment. Group allocation was performed using random number assignment, resulting in 62 patients being assigned to traditional treatment (Group A), 38 patients to APG treatment (Group B), and 55 patients to Huangma Ding treatment (Group C). There were no dropouts from the study. Both self-prepared Huangma Ding and APG application have been documented in the literature. Dressings were changed once a week[9,10]. The group assignments were kept undisclosed to the evaluators, researchers, and patients to maintain blinding throughout the study.

Method

Using the ICSHIB standard protocol, we conducted measurements for age, height, weight, BMI, blood pressure, and disease duration. Certified laboratories performed tests for fasting blood glucose (FPG), glycosylated hemoglobin (HbA1c), fasting insulin, HOMA-IR, total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein (HDL) levels. The intra-assay and interassay coefficients of variation for all parameters were < 5%. The Huntleigh MD2 Diabetes Screening Diagnostic Kit was utilized for ABI evaluation. Additionally, the transcutaneous oxygen pressure (TcPO₂) test utilized a TCM400 multichannel TcPO₂ analyzer as a reference. The primary treatment plan encompassed blood sugar control (insulin or oral hypoglycemic drugs), antibiotic therapy, surgical debridement, and drainage (removal of necrotic tissue and drainage without minor or major amputation), improvement of microcirculation (alprostadil), nutritional nerve therapy (methylcobalamin and alpha-lipoic acid), and supportive care. All patients received primary care. The orthopedic surgeon made decisions regarding the necessity and extent of amputation based on the patient’s preferences. Diabetic foot function was evaluated using the wound, ischemia, foot infection classification. Additionally, we employed the Armstrong and Frankberg classifications to categorize the surgical type into levels 1-4. These assessments and classifications were documented at the initial assessment.

Six months post-discharge, follow-up visits were conducted at an outpatient clinic. During these appointments, we gathered information on the DFU recovery rate (wound healing rate), amputation frequency, recurrence rate, ABI, TcPO₂, hospitalization frequency, and any other potential adverse events.

Safety precautions encompassed monitoring for adverse events such as postoperative infection, temporary fever, anaphylaxis, postoperative pain, postoperative bleeding, and patient-reported hypoglycemic episodes. Significant adverse events were characterized as those resulting in considerable morbidity, all-cause mortality, hospitalization, amputation, disability, or medical or surgical intervention to avert any of the other outcomes, in addition to those causing unacceptable disruption to the patient’s everyday activities.

Data collection and outcome measurement

The main outcomes assessed were the rates of amputation, both major (above the ankle) and minor (below the ankle), as well as wound healing rates at the end of six months of follow-up. Complete wound healing was defined as full re-epithelialization. Wounds that resulted in major amputations were categorized as unhealed. Conversely, wounds that healed following minor amputation were considered healed. Ulcers that reappeared after initial healing were classified as healed but were noted under the secondary outcome of ulcer recurrence. Patients with DFUs who were readmitted after their initial healing were categorized as rehospitalized.

The supplementary outcomes encompassed the ABI and TcPO₂ following a 6-month follow-up, absence of planned additional index finger revascularization at study commencement, new or recurring ulcers, and serious adverse events, such as substantial morbidity, all-cause mortality, ulcer recurrence, and readmission.

Statistical analysis

Statistical analysis was conducted using SPSS 19.0 software. A significance level of P < 0.05 was adopted. The data are presented as mean ± SD. Prior to analysis, normal distribution of the data was assessed using the Kolmogorov-Smirnov test. Group differences were examined using analysis of variance (ANOVA). Relationships between baseline metabolic and endocrine parameters and changes post-treatment were analyzed using a simple correlation approach. Pearson’s method was utilized to calculate variable correlations, covariates’ effects were adjusted through logistic regression, and independent factors were evaluated.

RESULTS

Baseline comparison of clinical data among the three groups

There were no significant differences in age; FPG, HbA1c, TC, TG, LDL-C, HDL, U.A., white blood cell (WBC), neutrophil, or blood pressure levels among the three groups (Table 1).

Table 1 Baseline characteristics of patients in each group (mean ± SD).

Parameter	Group A	Group B	Group C	P value
n	62	38	55
Age (yr)	60.83 ± 11.28	63.26 ± 10.94	62.89 ± 7.71	0. 401
Duration (yr)	9.81 ± 8.66	9.34 ± 6.68	7.31 ± 5.28	0.148
Weight (kg)	61.28 ± 7.47	60.60 ± 10.90	58.33 ± 8.52	0.178
Height (m)	1.63 ± 0.07	1.60 ± 0.10	1.61 ± 0.06	0.255
FPG (mmol/L)	8.45 ± 2.21	7.38 ± 3.18	8.40 ± 4.25	0.239
White blood cells	8.34 ± 2.66	7.51 ± 1.42	8.24 ± 2.30	0.181
Neutrophils	5.67 ± 2.61	4.85 ± 1.38	5.65 ± 1.99	0.134
HbA1c (%)	8.46 ± 1.66	8.00 ± 1.52	8.61 ± 1.71	0.203
LDL (mmol/L)	2.60 ± 0.70	2.56 ± 0.57	2.56 ± 0.52	0.932
HDL (mmol/L)	1.00 ± 0.30	1.10 ± 0.39	1.00 ± 0.18	0.240
TC (mmol/L)	4.62 ± 1.00	4.45 ± 0.97	4.58 ± 0.81	0.661
TG (mmol/L)	1.38 ± 0.52	1.49 ± 0.68	1.58 ± 0.56	0.233
Creatinine (mmol/L)	78.73 ± 18.62	84.69 ± 26.02	81.03 ± 20.36	0.398
History of smoking (yr)	7.45 ± 11.73	10.47 ± 16.43	7.76 ± 15.00	0.552
Hypertension Duration (yr)	5.11 ± 9.01	3.74 ± 5.97	3.53 ± 4.72	0.425
Uric acid (µmol/L)	308.61 ± 83.34	324.05 ± 98.75	338.05 ± 101.20	0.240
Diabetic retinopathy	24.19%	18.42%	16.36%	0.555
Diabetic peripheral neuropathy	96.77%	86.84%	87.27%	0.121
Diabetic kidney disease	41.94%	39.47%	50.91%	0.485
Coronary heart disease	9.68%	2.63%	1.27%	0.245
PAD	100%	100%	100%	1.000
Metformin	93.55%	89.47%	83.64%	0.233
Insulin secretagogues	14.52%	10.53%	18.18%	0.596
Acarbose	25.81%	23.68%	16.36%	0.453
Insulin	83.87%	86.84%	90.91%	0.530
ACEI/ARB	40.32%	47.37%	56.36%	0.225
CCB	27.42%	26.32%	27.27%	0.992
Diuretic	3.23%	7.89%	1.82%	0.314
SBP (mmHg)	134.01 ± 17.86	131.82 ± 16.67	137.84 ± 13.52	0.187
DBP (mmHg)	80.71 ± 9.49	78.90 ± 10.37	78.65 ± 10.84	0.503

FPG: Fasting blood glucose; HbA1c: Glycosylated hemoglobin; LDL: Low-density lipoprotein; HDL: High-density lipoprotein; TC: Total cholesterol; TG: Triglyceride; PAD: Peripheral artery disease; ACEI/ARB: Angiotensin-converting enzyme inhibitors/angiotensin II receptor blocker; CCB: Calcium channel blocker; SBP: Systolic blood pressure; DBP: Diastolic blood pressure.

The parameters before and after treatment were compared

Group A and Group B had significant differences (P < 0.05) in WBC count, neutrophil count, procalcitonin (PCT), TcPO₂, cardiovascular events, total number of amputations, readmission rate, reamputation rate, central amputation rate, minor amputation rate, healing rate, improvement rate, ulcer area, and diabetic foot classification. The number of WBCs in Group B decreased during follow-up (Table 2).

Table 2 Changes in parameters after each treatment (mean ± SD).

Parameter	Group A	Group B	Group C	P value
FPG (mmol/L)	6.37 ± 1.43	6.40 ± 1.26	6.14 ± 1.20	0.553
White blood cells	8.84 ± 3.46	7.35 ± 1.741	5.83 ± 1.352	0.000
Neutrophils	6.38 ± 3.66	4.97 ± 2.001	3.10 ± 1.122	0.000
Uric acid (µmol/L)	298.74 ± 71.33	294.61 ± 72.68	282.76 ± 63.40	0.444
SBP (mmHg)	126.37 ± 9.39	123.07 ± 10.60	124.27 ± 11.15	0.272
DBP (mmHg)	75.23 ± 5.84	73.76 ± 6.50	73.91 ± 4.56	0.325
PCT (mmol/L)	0.72 ± 0.95	0.36 ± 0.451	0.05 ± 0.072	0.000
HbA1c (%)	6.74 ± 0.87	6.76 ± 0.72	6.81 ± 0.52	0.855
LDL (mmol/L)	2.29 ± 0.48	2.27 ± 0.47	2.44 ± 0.46	0.141
HDL (mmol/L)	0.83 ± 0.26	0.74 ± 0.24	0.77 ± 0.22	0.136
TG (mmol/L)	2.63 ± 0.70	2.51 ± 0.82	2.57 ± 0.56	0.683
TC (mmol/L)	4.47 ± 0.53	4.49 ± 0.70	4.43 ± 0.62	0.901
ABI	0.76 ± 0.09	0.79 ± 0.08	0.81 ± 0.082	0.032
TcPO2 (mmHg)	35.56 ± 10.13	46.32 ± 8.831	48.19 ± 8.352	0.000
Total amputations	1.23 ± 1.14	0.63 ± 0.631	0.16 ± 0.372	0.000
Mortality rate	4.84%	0.00%	0.00%	0.102
Cardiovascular event rate	8.06%	0.00%1	0.00%2	0.020
Hospitalization rate	62.90%	26.32%1	5.45%2	0.000
Reamputation rate	58.06%	28.95%1	1.82%2	0.000
Major amputation rate	69.35%	31.58%1	36.40%2	0.000
Minor amputation rate	53.23%	31.58%1	14.55%2	0.000
Heal rate	14.52%	65.79%1	89.09%2	0.000
Improve rate	85.48%	36.84%1	10.91%2	0.000
Ulcer area (cm2)	3.74 ± 2.04	2.68 ± 1.961	1.77 ± 1.212	0.000
Diabetic foot surgery class	3.01 ± 0.64	2.08 ± 0.921	1.49 ± 0.962	0.000

¹Group A vs group B: P < 0.05.

²Group A vs group C: P < 0.05.

FPG: Fasting blood glucose; HbA1c: Glycosylated hemoglobin; LDL: Low-density lipoprotein; HDL: High-density lipoprotein; TC: Total cholesterol; TG: Triglyceride; CCB: Calcium channel blocker; SBP: Systolic blood pressure; DBP: Diastolic blood pressure; ABI: Ankle-brachial pressure; TcPO₂: Transcutaneous oxygen pressure; PCT: Procalcitonin.

Group A and Group C had significant differences (P < 0.05) in WBC count, neutrophil count, PCT, ABI, TcPO₂, cardiovascular events, total number of amputations, readmission rate, reamputation rate, central amputation rate, minor amputation rate, healing rate; improvement rate, ulcer area, and diabetic foot classification. The numbers of WBCs and neutrophils in Group C decreased over the course of the follow-up.

Significant differences (P < 0.05) between Group B and Group C were found in the ulcer area, diabetic foot grade, rehospitalization rate, reamputation rate, total amputation times, central amputation rate, healing rate, improvement rate, WBCs, neutral granulocytes, and procalcitonin level. Similarly, the ulcer area, diabetic foot grade, reamputation rate, rehospitalization rate, total amputation times, and improvement rate were lower in Group C, while the primary amputation rate was slightly greater in Group C. The ABI of both groups improved compared with that of the control group, but there was no significant difference between Groups B and C. There were no significant differences in TcPO₂ or the amputation rate between Groups B and C, but they both improved compared to those of the control group.

In conclusion, there was no significant difference in the primary data of the three groups before treatment. After treatment, the number of WBCs, number of neutrophils, and PCT improved in each group, although PCT was significantly better in Group C than in Group B. This finding shows that the anti-infection effect of Huangma Ding was better than that of basic treatment and APG.

According to the primary endpoint analysis, the major amputation rate, minor amputation rate, and total amputation times in Groups B and C were lower than those in Group A. These findings show that, compared with traditional treatment, the two treatment methods improved the macrocirculation and microcirculation of the lower extremity vessels. The ABI and TcPO₂ in Groups B and C were greater than those in Group A, and the diabetic foot classification was lower in both Groups B and C than in Group A, which further supports the efficacy of the two treatments. The total number of amputations in Group C was less than that in Group B, although Group C had a slightly greater rate of major amputation. There was no significant difference in the ABI between Groups B and C. Groups B and C had similar minor amputation rates. Taken together, these findings show that Huangma Ding did not play a substantial role in the whole circulation process at the beginning because microcirculation improvement combined with local anti-infection therapy improved the wider circulation and did not continue extensive amputation treatment. In addition, the readmission rate, reamputation rate, ulcer area, and diabetic foot grade in Group C were significantly lower than those in Group B, which further confirmed that after treatment in the later stage, the macrocirculation and microcirculation were significantly improved, and the infection was controlled. Moreover, the progression of the disease was controlled. In summary, the treatments in Groups B and C improved macrocirculation, microcirculation, and infection, and the treatment plan for Group C was better than that for Group B.

Cardiovascular events, rehospitalization rates, and reamputation rates, the secondary endpoints, were also signi-ficantly improved by the two treatments.

The relationship between the changes in each parameter six months after treatment

The total amputation rate was positively correlated with WBC count (r = 0.785, P < 0.05), neutrophil count (r = 0.779, P < 0.05), PCT level (r = 0.790, P < 0.05), ulcer area (r = 0.261, P < 0.05) and 0.05), readmission rate (r = 0.726, P < 0.05), reamputation rate (r = 0.765, P < 0.05), diabetes surgery classification (r = 0.309, P < 0.05), and major amputation rate (r = 0.852). The total amputation rate was negatively correlated with the ABI (r = -0.646, P < 0.05), TcPO₂ (r = - 0.650, P < 0.05), and healing rate (r = -0.436, P < 0.05) (Table 3).

Table 3 Relationships of all the parameters with the amputation rates after treatment.

Parameter	Total amputation	Major amputation	Minor amputation
R index
FPG	-0.006	0.029	-0.053
White blood cells	0.785a	0.709a	0.566a
Neutrophils	0.779a	0.699a	0.567a
Uric acid	0.094	0.099	0.06
SBP	0.102	0.072	0.085
DBP	0.1	0.102	0.063
PCT	0.790a	0.713a	0.577a
HbA1c	0.042	-0.002	0.081
LDL	-0.203
HDL	0.101	0.091	0.07
TG	0.06	0.008	0.098
TC	-0.041	-0.085	0.013
ABI	-0.646a	-0.516a	-0.552a
TcPO2	-0.650a	-0.537a	-0.528a
Ulcer area	0.261a	0.304a	0.103
Mortality rate	-0.155
Cardiovascular event rate	0.019	0.01	0.12
Hospitalization rate	0.726a	0.644a	0.542a
Reamputation rate	0.765a	0.678a	0.572a
Diabetic foot surgery class	0.309a	0.334a	0.15
Major amputation rate	0.852a	1	0.357a
Minor amputation rate	0.787a	0.357a	1
Heal rate	-0.436a	-0.355a	-0.378a
Total number of amputations	1	0.852a	0.787a
Improve rate	0.426a	0.347a	0.370a

^aP < 0.05.

FPG: Fasting blood glucose; HbA1c: Glycosylated hemoglobin; LDL: Low-density lipoprotein; HDL: High-density lipoprotein; TC: Total cholesterol; TG: Triglyceride; CCB: Calcium channel blocker; SBP: Systolic blood pressure; DBP: Diastolic blood pressure; ABI: Ankle-brachial pressure; TcPO₂: Transcutaneous oxygen pressure; PCT: Procalcitonin.

The major amputation rate was positively correlated with WBC count (r = 0.709, P < 0.05), neutrophil count (r = 0.699, P < 0.05), PCT level (r = 0.713, P < 0.05), ulcer area (r = 0.304, P < 0.05) and 0.05), readmission rate (r = 0.644, P < 0.05), reamputation rate (r = 0.678, P < 0.05), diabetes surgery classification (r = 0.334, P < 0.05), minor amputation rate (r = 0.357, P < 0.05), total number of amputations (r = 0.852, P < 0.05), and improvement rate (r = 0.347, P < 0.05). The major amputation rate was negatively correlated with the ABI (r = -0.516, P < 0.05), TcPO₂ (r = - 0.537, P < 0.05), and healing rate (r = -0.355, P < 0.05).

The minor amputation rate was positively correlated with the WBC count (r = 0.566, P < 0.05), neutrophil count (r = 0.567, P < 0.05), PCT level (r = 0.577, P < 0.05), readmission rate (r = 0.542, P < 0.05), reamputation rate (r = 0.572, P < 0.05), major amputation rate (r = 0.357, P < 0.05), total number of amputations (r = 0.787, P < 0.05), and improvement rate (r = 0.370, P < 0.05), while it was negatively correlated with the ABI (r = -0.552, P < 0.05), TcPO₂ (r = -0.528, P < 0.05), and healing rate (r = -0.378, P < 0.05).

Logistic regression analysis of each parameter after treatment

The total amputation rate was positively correlated with the WBC count and PCT and negatively correlated with the ABI and TcPO₂. The strengths of the correlations were ordered as follows: TcPO₂ > ABI > PCT > WBC count (Tables 4-6).

Table 4 Logistic regression analyses of total amputation after treatment.

Total amputation	B	S.E.	df	P value	95% confidence interval	OR
Neutrophils	0.003	0.042	1.000	0.936	-0.886	0.080
White
ABI	0.081	0.043	1.000	0.064	-0.171	1.867
TcPO2	-2.407	0.455	1.000	0.000	-2.251	-5.285
PCT	-0.023	0.004	1.000	0.000	-0.015	-6.157
	0.458	0.096	1.000	0.000	0.268-0.648	4.760

The total amputation rate was positively correlated with the white blood cell count and procalcitonin (PCT) and negatively correlated with the ankle-brachial pressure (ABI) and transcutaneous oxygen pressure (TcPO₂). The strengths of the correlations were ordered as follows: TcPO₂ > ABI > PCT > white blood cell count. OR: Odd ratio; ABI: Ankle-brachial pressure; TcPO₂: Transcutaneous oxygen pressure; PCT: Procalcitonin.

Table 5 Logistic regression analyses of major amputation after treatment.

Major amputation	B	S.E.	df	P value	95% confidence interval	OR
ABI	-0.892	0.411	1.000	0.032	-1.624	-2.170
TcPO2	-0.011	0.003	1.000	0.001	0.062	-3.277
Neutrophils	-0.005	0.038	1.000	0.896	-0.150	-0.131
White	0.061	0.039	1.000	0.119	-0.154	1.570
PCT	0.293	0.087	1.000	0.001	0.122-0.465	3.378

Major amputation was positively correlated with procalcitonin (PCT) and negatively correlated with the ankle-brachial pressure (ABI) and transcutaneous oxygen pressure (TcPO₂), and the order of significance was PCT > TcPO₂ > the ABI. OR: Odd ratio; ABI: Ankle-brachial pressure; TcPO₂: Transcutaneous oxygen pressure; PCT: Procalcitonin.

Table 6 Logistic regression analyses of minor amputation after treatment.

Minor amputation	B	S.E.	df	P value	95% confidence interval	OR
ABI	-1.558	0.411	1.000	0.000	-3.117	-3.791
TcPO2
Neutrophils	-0.012	0.003	1.000	0.001	-0.024	-3.470
White	0.008	0.038	1.000	0.843	-0.149	0.198
PCT	0.015	0.039	1.000	0.696	-0.159	0.391
	0.177	0.087	1.000	0.044	0.005-0.348	2.033

Minor amputation was positively correlated with procalcitonin and negatively correlated with the ankle-brachial pressure (ABI) and transcutaneous oxygen pressure (TcPO₂), with the correlation strengths of ABI and TcPO₂ > procalcitonin. OR: Odd ratio; ABI: Ankle-brachial pressure; TcPO₂: Transcutaneous oxygen pressure; PCT: Procalcitonin.

Major amputation was positively correlated with PCT and negatively correlated with the ABI and TcPO₂, and the order of significance was PCT > TcPO₂ > the ABI.

Minor amputation was positively correlated with PCT and negatively correlated with the ABI and TcPO₂, with the correlation strengths of ABI and TcPO₂ > PCT.

DISCUSSION

In diabetic patients, peripheral arterial disease typically presents as widespread and is characterized by long-segment arterial occlusion rather than stenosis. This condition is distinguished by the presence of medial arterial calcification, a common feature of intraluminal atherosclerosis in peripheral arterial disease among non-diabetic individuals[11,12]. The prolonged high blood sugar levels in diabetes have a lasting detrimental effect on the arteries, major blood vessels, and microvessels of the lower limbs. Vascular complications linked to diabetic peripheral arteries cause reduced blood flow to infected tissues, leading to hypoperfusion. The presence of multiple neuropathies increases the susceptibility to diabetic foot infections. In cases of DFUs, the normal wound repair process is disrupted, failing to progress through the typical stages of inflammation, angiogenesis, neovascularization, and extracellular matrix remodeling required for dermal repair. As a result, DFU wound healing becomes stalled during the inflammatory phase, resulting in chronic wounds. Despite receiving antibiotic treatment, one study found that 12.7% (40 out of 314) of patients with DFUs died, and an additional 24.5% (77 out of 314) required lower extremity amputation. For severe infections unresponsive to antibiotics, surgical revascularization becomes necessary[12].

In our prior cohort study, we observed that interventional surgery could effectively restore patency in major blood vessels for patients with DFUs and lower extremity ischemia. However, the primary clinical benefit of this procedure was a reduction in the incidence of major amputations, with minor amputation cases showing only modest enhancements in outcomes. APG, a secondary platelet-derived agent, has emerged as a valuable supplementary approach for managing acute or chronic wounds. These cytokines, comprising platelets, leukocytes, fibrin, growth factors, and cytokines, exhibit anti-infective properties[13] and play a role in immunoregulation[14]. Leukocytes influence the generation and release of growth factors, including transforming growth factors, which stimulate angiogenesis. Fibrin is crucial in the action of vascular endothelial growth factor APG and can enhance healing by promoting adhesion and fibronectin effects. Application of APG uniformly on a wound or skin ulcer resulting from platelet rupture leads to the formation of a high-concentration growth factor layer that aids in wound healing. Moreover, platelets and the activation process release active antimicrobial peptides to combat microorganisms and prevent wound infections[15,16]. Huangma Ding is a traditional Chinese medicine formula designed for topical application. It comprises Nuxychnium and the Coptidium Rhizome. While Nuxychnium can be toxic if consumed, it poses no harm when used externally. The primary active compound found in this mixture is strychnine, which is known for its high toxicity. However, after dissolving Qianzi in alcohol, the remaining component is strychnine, which exhibits anti-inflammatory and muscle relaxant properties. Clinical reports suggest that this product demonstrates significant efficacy in combating Pseudomonas aeruginosa, certain fungi, and various types of cocci[17]. Within Coptidium, berberine is extracted using ethanol and serves as a wide-ranging aminoglycoside medication. Berberine displays anti-inflammatory properties against gram-negative bacteria, showcasing bacteriostatic or bactericidal effects. Additionally, berberine is noted for its muscle relaxant capabilities[18,19]. Berberine acts as a natural PPARγ agonist, and PPARγ plays a role in the endothelial signaling pathway. This signaling pathway involving the vascular endothelial growth factor facilitates the promotion of microvessel re-construction[8,20]. We hypothesize that Huangma Ding may enhance wound healing by exerting anti-inflammatory effects and enhancing local blood and oxygen supply to wounds. In our previous research, our team observed that Huangma Ding and similar topical medications could enhance the healing process of DFUs and decrease the amputation rate by addressing local ischemia and hypoxia[8,21].

The initial infection status was consistent among the three groups prior to treatment. Both the APG and Huangma Ding therapeutic approaches demonstrated superior efficacy compared to the conventional treatment method, with Huangma Ding exhibiting a more pronounced anti-infection effect than APG. These results indicate that both Huangma Ding and APG, particularly Huangma Ding, possess notable local anti-infection properties.

In prior research, our team discovered that Huangma Ding and comparable topical medications enhanced the healing rate of DFUs and lowered the amputation rate by addressing local ischemia, hypoxia, and inflammation. Additionally, it improved macrocirculation, potentially aiding in promoting DFU healing by enhancing the local inflammatory response and microcirculation, thereby further facilitating the improvement of macrocirculation. APG delivers a range of growth factors and cytokines, enhances microcirculatory blood supply, improves local inflammation and immune function, suppresses bacterial growth, promotes wound healing, and enhances clinical outcomes. While previous studies have suggested that both APG and Huangma Ding possess anti-infective properties, our findings indicate that Huangma Ding exhibits a superior anti-infective effect compared to APG. As previously mentioned, berberine and strychnine, derived from Coptis chinensis and Nux vomica, respectively, contribute to the anti-infective effects of Huangma Ding. Based on our research, it is reasonable to infer that Huangma Ding demonstrates a more pronounced anti-infective effect than APG. Furthermore, local anti-infection treatment plays a crucial role in promoting wound healing in diabetic foot wounds.

Further logistic regression analysis indicated a positive correlation between PCT levels and total, major, and minor amputations. Conversely, ABI and TcPO₂ demonstrated a negative correlation with total, major, and minor amputations. These results underscore the significance of local infection, macrocirculation, and microcirculation in determining diabetic foot outcomes, regardless of the type of amputation. The odds ratios (ORs) for extensive amputations were 3.378 for PCT, -3.277 for TcPO₂, and -2.170 for ABI, while the ORs for minor amputations were -3.791 for ABI, -3.470 for TcPO₂, and 2.033 for PCT. Effective infection management is also crucial for enhancing the prognosis of major amputations.

In our earlier study, we found that major amputation was primarily associated with the ABI, while minor amputation was predominantly linked to the local TcPO₂ index. In this current research, both the TcPO₂ levels and rates of minor amputations were lower in the Huangma Ding and APG cohorts compared to the traditional treatment group. Furthermore, there was no significant variance between the two intervention groups. The ABI values decreased in both treatment cohorts in contrast to the traditional treatment group, yet there was no notable distinction between the two treatment modalities. The initial amputation rate in both treatment cohorts was lower than that in the control group, but it was slightly higher in the Huangma Ding group compared to the APG group. Subsequent analysis indicated that the rehospitalization rate, reamputation rate, ulcer area, and diabetic foot classification were significantly lower in the Huangma Ding group than in the APG group. Initially, it appeared that Huangma Ding did not exert a significant impact on macrocirculation. However, subsequent treatments led to improvements in microcirculation, while local anti-infection therapy gradually enhanced macrocirculation. As a result, primary amputation was avoided, leading to reductions in readmission rates, reamputation rates, and ulcer areas. The grade of DFUs was also lowered with the use of Huangma Ding. Although neither Huangma Ding nor APG directly influenced macrocirculation, this study demonstrated a significant decrease in PCT levels in both groups. Notably, the reduction in PCT levels with Huangma Ding was substantially greater than with autologous platelet gel. Both treatment groups showed enhanced microcirculation and local inflammatory responses, facilitating the healing of DFUs and subsequent improvements in macrocirculation. This observation indicates that in patients with DFUs and concurrent infections, managing infection is equally crucial to enhancing circulation.

CONCLUSION

In conclusion, patients with DFUs can experience reduced rates of major amputation, minor amputation, and total amputation through local anti-infection therapy and enhanced microcirculation and macrocirculation. Both Huangma Ding and APG demonstrate effectiveness in treating DFUs, with Huangma Ding exhibiting superior clinical efficacy compared to APG, possibly due to its stronger capacity to manage local infection.

ARTICLE HIGHLIGHTS

Research background

Diabetes foot is one of the most serious complications of diabetes and an important cause of death and disability. Traditional treatment has poor efficacy and there is an urgent need to develop practical treatment methods.

Research motivation

Our previous study found that the addition of Huangma ding can significantly improve the healing rate of diabetes foot ulcers, but the mechanism is unclear. To investigate the role and mechanism of Huangma ding in promoting wound healing of diabetes foot ulcers, we conducted this study.

Research objectives

To investigate whether Huangma Ding or autologous platelet-rich gel (APG) treatment would benefit diabetic lower extremity arterial disease (LEAD) patients with foot ulcers.

Research methods

Diabetic LEAD patients with foot ulcers were enrolled and divided into three groups. All patients underwent routine follow-up visits for six months. We calculated the changes in all variables from baseline and determined the differences.

Research results

Our research indicated that diabetic foot ulcers (DFUs) lead to major amputation, minor amputation, and total amputation through local infection and poor microcirculation and macrocirculation.

Research conclusions

Huangma Ding and APG were effective at treating DFUs. The clinical efficacy of Huangma Ding was better than that of autologous platelet gel, which may be related to the better control of local infection by Huangma Ding. In DFUs caused by coinfection, controlling infection is as important as improving circulation.

Research perspectives

The clinical efficacy of Huangma Ding was better than that of autologous platelet gel, which may be related to the better control of local infection by Huangma Ding.