Efficacy of low-dose insulin combined with electrolyte in the treatment of pediatric diabetic ketoacidosis and its effect on serum inflammatory factors

: Diabetic ketoacidosis (DKA) is a very serious disease that can occur in both types of diabetes (type 1 and 2). It is caused by a combination of high blood sugar and low insulin levels, which can cause the body to produce too much ketone. Ketones are toxic to human organs. This research aimed to investigate the clinical efficacy of low-dose insulin combined with electrolyte in the treatment of pediatric DKA and its effect on serum inflammatory factors. For this purpose, a total of 122 children with DKA admitted to our hospital from April 2013 to May 2016 were selected as research objects. They were divided into group A with 60 cases and group B with 62 cases. Group B was treated with supplemental electrolytes, and group A was treated with low-dose insulin based on group B. The serum levels of TNF-α, IL-6, and IL-18 were measured by enzyme-linked immunosorbent assay (ELISA) before and after treatment, and the blood sugar, sodium, and potassium levels were measured by an automatic biochemical analyzer. The time when blood sugar reached the standard level when acidosis was corrected and hospitalization time was compared between the two groups. The total effective rate of group A was significantly higher than that of group B ( p < 0.05). There was no significant difference in blood glucose, sodium, potassium, TNF-α, IL-6, and IL-18 levels between the two groups before treatment. (all p > 0.05). But the blood glucose, sodium and potassium levels in group A were significantly better than those in group B (all p < 0.001). The levels of serum TNF-α, IL-6, and IL-18 in group A were significantly lower than those in group B after treatment (all p < 0.001). After treatment, the time when blood sugar reached the standard level when acidosis was corrected and hospitalization time in group A were significantly shorter than those in group B (all p < 0.001). Low-dose insulin combined with electrolyte supplementation is effective in the treatment of DKA in children, which can effectively control blood sugar, sodium, potassium level, and inflammatory factor concentration.


Introduction
Diabetic ketoacidosis (DKA) is an acute complication of diabetes. This complication is characterized by three biochemical characteristics: ketosis, acidosis and hyperglycemia. The DKA may be an early sign of type 1 diabetes, but it is usually when the level of insulin in the blood is lower than the body's needs (for example, forgetting the insulin dose or increasing the insulin dose under stressful conditions such as illness Bottom) diagnosed with diabetes and surgery. The DKA is characterized by shortness of breath, shortness of breath with ketone smell, nausea and vomiting, dehydration and abdominal pain or decreased consciousness. Treatment is a specialized treatment that usually requires emergency care in the intensive care unit. The main methods of treatment are hydration and control of sugar and acidosis (1)(2)(3)(4)(5). The DKA is a common disease in pediatrics and is a serious complication caused by diabetes in children. It is mainly caused by severe insufficiency of insulin and abnormal increase of blood sugar hormone, leading to metabolic disorders of related substances (1,2). The main symptoms of the disease are nausea, vomiting, po-lyuria, dehydration, and abdominal pain, which is characterized by rapid progress and sudden illness (3). If not timely treated, the children will be comatose or even dead (4). Therefore, exploring the appropriate treatment of DKA has always been a hot spot in clinical research (5,6).
In clinical practice, due to electrolyte disturbance and dehydration in children, timely supplementation of electrolytes not only greatly improves renal function and blood circulation function, but also accelerates the discharge of acidosis substances into the body with urine, and at the same time, better reduce blood sugar levels (7). Previous treatment of DKA in the clinic often adopted the methods of active and effective fluid replacement and insulin injection. Because the blood osmotic pressure drops suddenly after high dose injection, complications such as hypoglycemia and brain edema often occur. Therefore, in recent years, the use of low-dose insulin injection gradually replaces high dose insulin injection, and the effect is satisfactory (8,9). Recent studies have shown that increased levels of inflammatory factors such as TNF-α, IL-6, and IL-18 have also been detected in DKA patients (10)(11)(12). Although the mecha-nism is not yet clear, it suggests that inflammation is closely related to the occurrence and development of diabetes mellitus, even inflammatory cytokines may play an important role in the development of DKA.
Clinical treatment of DKA children mainly aims to correct electrolyte disorder, acid-base imbalance, and hypoglycemia (13). However, there is little treatment for DKA children with low-dose insulin combined with electrolyte. In this study, low-dose insulin was given while electrolyte supplementation was used in DKA children. The changes of inflammatory factors TNF-α, IL-18, and IL-6 were observed in the treatment, so as to provide a reference for the treatment of children with DKA.

General information
A total of 122 children with DKA admitted to our hospital from April 2013 to May 2016 were selected as study subjects. They were divided into group A (60 patients) and group B (62 patients). The two groups were treated with supplemental electrolytes. Based on this, group A was treated with low-dose insulin. Group B was treated with large dose insulin, and 0.1-0.20 U/ (kg·h) of insulin was given to patients with a subcutaneous injection before bedtime, once a day. There were 32 males and 28 females in group A, aged 1 to 11 years, with an average age of 6.19 ± 1.86 years; 36 males and 26 females in group B, aged from 2 to 11 years, with an average age of 6.37 ± 1.52 years. Inclusion criteria: All children were diagnosed in accordance with the diagnostic criteria for the Guidelines for Diagnosis and Treatment of DKA in Children; their age was less than 12 years old; this study was not against ethics and morality, and the program was submitted to the Hospital Ethics Committee for review and approval; the guardians of children voluntarily signed the informed consent document. Exclusion criteria: patients with cerebral edema, and septic shock; 6 hours without urine; accepted insulin therapy before admission; patients with severe liver and kidney dysfunction, malignant tumor; patients with congenital heart disease, congenital neuropathy, dilated cardiomyopathy, heart failure, cardiogenic shock and malignant tumors.

Treatment methods
Two groups of children were treated with symptomatic treatment and supportive treatment after admission. Group B was treated with electrolyte supplementation. After admission, the children were given routine tests and oxygen inhalation, and electrolyte, blood sugar, and blood gas experiments in time. The severity of acidosis and dehydration was evaluated scientifically, and symptomatic treatment was given in time. For children with moderate or severe DKA, intravenous channels should be established in time and physiological saline should be completely given within 60min with a dose of 10-20 ml/kg. When the peripheral circulation is poor, repeated treatment is required. Sodium chloride with 0.45% concentration at the initial speed of 10 mL (kg. H) was given immediately. After 60-120 minutes of installation, the speed was adjusted to 5 mL(kg·h), and the drip rate was adjusted based on the physical condition.
When there is no contraindication of potassium solution in children, potassium solution should be added to normal saline and sodium chloride. On this basis, the second channel was established to drip insulin (Jiangsu Wanbang Biochemical and Pharmaceutical Co., Ltd., batch number: H32024567). In group A, insulin was given 0.1 U (kg/·h); in group B, insulin was given 0.2 U (kg·h). If the blood sugar level was still above 15 mmol/L, the insulin input would be increased by 25%. If the blood sugar level was below 8 mmol/L, the sugar input concentration would be increased by 10% on the original basis, and the insulin infusion dose would be kept at 0.05 U/kg per hour.

Main instruments and reagents
TNF-α enzyme-linked immunosorbent assay (ELI-SA) kit was purchased from Shanghai Xiyuan Biological Technology Co., Ltd., China, batch number: XY-BGK01375. IL-18 and IL-6 ELISA diagnostic kits were purchased from Shanghai Guduo Biology Co., Ltd., China, with the numbers GD-G10207 and GD-G10211, respectively. A fully automatic biochemical analyzer is manufactured in Human, Germany, batch number: 16660. ELISA is manufactured in Molecular Devices, USA, batch number: SpectraMaxiD5.

Observation indicators
The corrective time of acidosis, the time of blood sugar reaching the standard, and the time of hospitalization were observed and recorded in the two groups. Blood glucose, serum potassium, serum potassium levels, serum inflammatory factors (IL-6, IL-18, TNF-α) were measured before and after treatment. Blood glucose, serum potassium, and potassium levels were detected by an automatic biochemical analyzer. Serum TNF-α, IL-6, and IL-18 concentrations were detected by ELISA: standard wells, sample wells, and blank control wells were set up (not add sample and enzymelabeled reagent), the enzyme label is accurately loaded with 50μl of the standard on the plate, and add 40μl of the sample diluent, then 10μl of the sample to be tested in the sample wells (the final dilution of the sample is 5 times). Then the wells were covered with the membrane, incubate at 37℃ for 30 min; discard the liquid in each well, pat dry, repeat washing 5 times; add 50μl of enzyme labeling reagent to each well except for blank wells, cover with a membrane, incubate at 37 ℃ for 30 min; add 50μl chromogenic reagent A to each well, then add 50μl developer B, mix and develop color at 37 ℃ for 10 min in the dark; add 50μl stop solution to each well to terminate the reaction. The absorbance (OD) of each hole was measured sequentially at 450nm wavelength by ELISA detector, and the concentration of IL-6, IL-18, and TNF-α was calculated.
Efficacy judgment are 1) markedly effective: the symptoms of the child basically disappeared, the ketone body turned negative, and the blood sugar returned to normal, 2) effective: the symptoms of the child improved significantly, the ketone body (+), and the blood sugar was slightly higher than the normal value, and 3) invalid: No significant change or even aggravation of symptoms and indicators in children. Significant efficiency + effective rate = the total efficiency. were markedly effective, 20 cases (32.26%) were effective, 16 cases (25.81%) were ineffective, and the total effective rate was 74.19% in group B. Obviously, the total effective rate in group A after treatment was significantly higher than that in group B (p< 0.05, Table 2).

Statistical methods
Statistical analysis was performed using SPSS 21.0 (IBM Corp, Armonk, NY, USA). Counting data were expressed by case/percentage (n (%)). A Chi-square test was used to compare the counting data between groups. Mean ± standard deviation (x±sd) was used to express the measurement data. An independent sample t-test was used to compare the measurement data between groups, but the paired t-test was used to compare before and after the same group. When p< 0.05, the difference was statistically significant.

Two groups of general information
There was no significant difference in gender, age, weight, height, weight, residence, parental alcoholism history, parental smoking history, ethnicity and maternal education level between group A and group B (all p> 0.05, Figure 1).

Comparison of clinical efficacy between group A and group B
After treatment, 32 cases (53.34%) were markedly effective, 23 cases (38.33%) were effective, 5 cases (8.33%) were ineffective, and the total effective rate was

Comparisons of acidosis correction time, blood glucose achievement time and hospitalization time between group A and group B
The time when acidosis was corrected, the time when blood sugar reached the standard level and the time of hospitalization in group A were significantly shorter than those in group B. There was a significant difference between the two groups based on Table 3 & Figure 1(all p< 0.05).
The time when acidosis was corrected in group A and group B was compared (A), and the correction time of acidosis in group A was significantly shorter than that in group B (p< 0.05). The time when blood sugar reached the standard of group A and group B was compared (B), and the time when blood sugar reached the standard of group A was significantly shorter than that of group B. The hospitalization time of group A and group B were compared (C), and the hospitalization time of group A was significantly shorter than that of group B.

Changes in blood glucose, serum potassium and blood sodium concentration before and after treatment in group A and group B
There were no significant differences in serum glucose, serum potassium, and serum sodium levels between group A and group B before treatment (all P> 0.05). The blood glucose and serum sodium levels in group A and group B were significantly lower than the pre-treatment levels (both p< 0.05), while the serum potassium levels were significantly higher than the pre-treatment levels (p< 0.05). After treatment, the blood glucose and serum sodium levels in group A were significantly lower than those in group B (both p< 0.05), while the serum potassium level was significantly higher than that in group B based on Table 4 & Figure 2 (p< 0.05).
Comparison of blood glucose levels before and after treatment in group A and group B (A), there was no significant difference in serum glucose levels between group A and group B before treatment (both P> 0.05). The blood glucose levels in group A and group B were significantly lower than those before treatment (both p< 0.05), and the blood glucose level in group A was significantly lower than that in group B after treatment (p< 0.05).
Comparison of serum potassium levels between group A and group B before and after treatment (B), there was no significant difference in serum and blood potassium levels between group A and group B before and after treatment (both P> 0.05). Serum and blood potassium levels in group A and group B after treatment were significantly higher than those before treatment (both p< 0.05), and serum potassium levels in group A after treatment were significantly higher than those in group B after treatment (p< 0.05).
Comparison of serum sodium levels between group A and group B before and after treatment (C), there was no significant difference in serum sodium levels between group A and group B before treatment (both P> 0.05). The blood sodium levels in group A and group B were significantly lower than those before treatment (both p< 0.05), and the blood sodium level of group A was significantly lower than that of group B after treatment (p< 0.05).

Changes of serum TNF-α, IL-6 and IL-18 concentrations before and after treatment in group A and group B
There were no significant differences in serum   Table 4. Comparison of blood glucose, serum potassium and blood sodium control indexes before and after treatment in group A and group B (x±sd).
Note: Compared with before treatment, * indicated p< 0.05. TNF-α, IL-6, and IL-18 concentrations between group A and group B before treatment (all P> 0.05). The concentrations of serum TNF-α, IL-6, and IL-18 in group A and group B were higher after treatment. The concentration of serum TNF-α, IL-6, and IL-18 in group A was significantly lower than that in group B after treatment (all p< 0.05). See Table 5, Figure 3.
The serum TNF-α concentration was compared between group A and group B before and after treatment (A), and there was no significant difference between group A and group B before treatment (both p> 0.05). After treatment, the serum TNF-α concentration in group A and group B decreased significantly (p <0.05). After treatment, the serum TNF-α concentration in group A was significantly lower than that in group B (p <0.05).
Serum IL-6 levels were compared between group A and group B before and after treatment (B). There was no significant difference in serum IL-6 concentration between group A and group B before treatment (all P> 0.05). Compared with before treatment, the serum IL-6 concentration in Group A and Group B was significantly reduced after treatment (both p <0.05). After treatment, the serum TNF-α concentration in group A was significantly lower than that in group B (p <0.05).
According to comparison of serum IL-18 concentration between group A and group B before and after treatment (C), there was no significant difference in serum IL-18 concentration between group A and group B before treatment (both p> 0.05). Compared with before treatment, the serum IL-18 concentration in group A and group B was significantly reduced after treatment (both P <0.05). After treatment, the serum IL-18 concentration in group A was significantly lower than that in group B (p <0.05).

Discussion
DKA is a common complication in children with diabetes mellitus, with increased incidence in recent years (14,15). The incidence of DKA in children is relatively acute. If the children can be effectively rescued in time in the early stage, and the blood sugar level of the children can be decreased timely, it can be ensured that the children can safely get through the danger period (16). Therefore, effective and in-place rescue is of great significance to the prognosis quality of children with DKA (17,18).
The key to the treatment of DKA in children is to correct acidosis, restore water, and electrolyte balance (19). In the fluid infusion and clinical practice, based on routine treatment such as correction of water and electrolyte disorders, insulin supplementation therapy for children can specifically inhibit the formation of ketone bodies and help patients effectively control the disease, and the clinical effect is remarkable (20,21). However, the grasp of insulin dose during treatment is very important (22). A study conducted by Cemeroglu et al. (23) has pointed out that adult guidelines are not suitable for children and that age should be taken into account when calculating insulin doses. Nallasamy (24) and others found that the efficacy of low-dose insulin in the treatment of children with DKA is not inferior to that of standard-dose in terms of blood sugar reduction rate and acidosis regression rate. In this study, children with DKA were treated with low-dose insulin combined with electrolyte supplementation. The results showed that the total effective rate of group A drugs was better than that of group B. After treatment, the time when blood sugar reached the standard level, the time when acidosis was corrected and the time of hospitalization in group A was significantly shorter than those in group B, and the control of blood sugar, sodium and potassium levels in group A was significantly better than that in group B. This suggests that supplementation of electrolytes in children with DKA or combination with low-dose insulin and electrolyte supplementation is a viable chemotherapy regimen, while low-dose insulin combined with electrolyte supplementation may be more effective in treating children with DKA.
Diabetes patients are often susceptible to secondary inflammatory reactions due to low self-resistance and elevated blood sugar, and infections are prone to acute complications such as DKA (25,26). Recent studies have found that DKA is associated with increased inflammatory factors in the body (27). IL-6, IL-18,  Table 5. Comparison of serum TNF-α, IL-6 and IL-18 levels (x±sd).
Note: Compared with before treatment, * indicated p< 0.05. and TNF-α are common inflammatory cytokines with multiple functions, which are considered to play an important role in DKA reaction (28)(29)(30). In this study, the concentrations of serum IL-18, IL-6, and TNF-α in group A and group B were significantly lower than those before treatment, and the serum levels of IL-18 and IL-6 in group A were significantly lower than those in group B after treatment. This suggests that low-dose insulin combined with electrolytes can improve the inflammatory response in children with DKA. In the study of Popovic et al. (31), the levels of inflammatory factors CRP and IL-6 in DKA patients after intravenous insulin therapy were significantly decreased, which is similar to our study. This suggests that low-dose insulin combined with electrolyte therapy is related to the alleviation of inflammatory response. Thus inhibiting the levels of inflammatory factors may be one of its therapeutic mechanisms (32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51).
In this study, the subjects were screened according to the exclusion and inclusion criteria. There was no significant difference in general clinical baseline data such as sex, age, and weight between groups A and B, which ensured the rigor and reliability of the study. It was confirmed that low-dose insulin combined with electrolyte supplementation had obvious benefits in the DKA application, but the risk factors of DKA in children were not observed in the study. The clinical and pathological parameters of DKA in children were different from those in other age stages. Whether low-dose insulin combined with electrolyte supplementation was suitable for DKA patients of other ages has not been explored. These shortcomings need to be further supplemented in future studies, and further evidence for the results of this study will be provided.
In conclusion, the low-dose insulin combined with electrolyte therapy is effective in children with DKA, and can effectively control blood glucose, sodium, potassium, and inflammatory cell levels, which is worthy of further clinical application.
Authors' contributions ML W,LH F and W Z conceived and designed the study, collected, analyzed and interpreted the experiment data, drafted this paper, and revised the manuscript critically for important intellectual content. Three authors read and approved the final manuscript.