Low-level laser irradiation effect on endothelial cells under ...

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Abstract

Diabetes mellitus is considered to be a very serious lifestyle disease leading to cardiovascular complications and impaired wound healing observed in the diabetic foot syndrome. Chronic hyperglycemia is the source of the endothelial activation. The inflammatory process in diabetes is associated with the secretion of inflammatory cytokines by endothelial cells, e.g., tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6). The method of phototherapy using laser beam of low power (LLLT&#;low-level laser therapy) effectively supports the conventional treatment of diabetic vascular complications such as diabetic foot syndrome. The aim of our study was to evaluate the effect of low-power laser irradiation at two wavelengths (635 and 830 nm) on the secretion of inflammatory factors (TNF-α and IL-6) by the endothelial cell culture&#;HUVEC line (human umbilical vein endothelial cell)&#;under conditions of hyperglycemia. It is considered that adverse effects of hyperglycemia on vascular endothelial cells may be corrected by the action of LLLT, especially with the wavelength of 830 nm. It leads to the reduction of TNF-α concentration in the supernatant and enhancement of cell proliferation. Endothelial cells play an important role in the pathogenesis of diabetes; however, a small number of studies evaluate an impact of LLLT on these cells under conditions of hyperglycemia. Further work on this subject is warranted.

Keywords:

Low-level laser therapy, TNF-alpha, IL-6, Endothelial cells

Introduction

In the twenty-first century, diabetes mellitus has become an epidemic and being a risk factor of cardiovascular diseases [1] leads to an increased mortality in the population of developed and developing countries. Its severe complication&#;diabetic foot syndrome&#;is the primary cause of limb amputation due to vascular complications. An impaired wound healing is crucial in the clinical manifestation of diabetes, and hyperglycemia in uncontrolled diabetes is the major pathogenic factor.

The process of wound repair involves several consecutive phases (inflammatory and proliferative phase, remodeling of the wound), and it demands various factors: polypeptide growth factors, cytokines, and extracellular matrix components [2, 3]. Many of them are derived from endothelial cells, namely vascular endothelial growth factor (VEGF), tissue plasminogen activator (t-PA), metalloproteinases (MMP-2, MMP-9) and their inhibitors, tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6). Therefore, the endothelium is essential in the maintenance of vascular homeostasis [4]. Hyperglycemia in diabetes is responsible for damaging of the endothelium and increases inflammation on the surface of the vascular lining [5].

TNF-α enhances the inflammation process and has an impact on angiogenesis and destructive processes. It induces the production of other pro-inflammatory cytokines, e.g., IL-6. It contributes to oxidative stress by generating reactive oxygen species (ROS) [6]. TNF-α is the most important pro-inflammatory factor in diabetes.

IL-6 is a pleiotropic cytokine with a wide spectrum of biological activity. Its main function is to participate in the immune response and acute phase of inflammatory reaction where it acts together with TNF-α [7]. In the acute inflammatory response, Il-6 has regulatory properties and limits the production of pro-inflammatory cytokines such as TNF-α [8]. IL-6 can therefore suppress the development of diabetes. However, when produced in excess and in long term, it promotes a passage of inflammation into chronic phase and contributes to the development of many diseases [9].

Many studies have shown that high glucose concentration (20&#;40 mM/L) in the culture medium imitates conditions as in uncontrolled diabetes and adversely affects cell proliferation leading to cell damage and apoptosis [10&#;12].

The number of patients suffering from diabetes is still increasing, and chronic complications of microangiopathy and macroangiopathy cause disability and inability to work and a deterioration of the quality of life. Diabetes is also the leading cause of amputation [13] due to non-healing wounds. The method of phototherapy using laser beam of low power (LLLT&#;low-level laser therapy) effectively supports conventional treatment and brings a significant improvement in the quality of life in diabetes.

According to Brosseau et al. [14], lasers have been used in medicine since at least when LLLT was officially implemented in the USSR as an alternative non-invasive treatment of rheumatoid arthritis. A number of studies have reported the positive impact of LLLT on alleviation symptoms caused by hyperglycemic conditions. These investigations involved humans and animals and were conducted in vitro [15&#;17] and in vivo [18&#;20]. Mainly, they evaluated fibroblasts and osteoblasts. Research from the last decade provides data on the key role of the vascular endothelium in maintaining vascular homeostasis and pathogenesis of vascular diseases. Endothelial cells line the walls of blood vessels and therefore are on the front line of contact with the high concentration of glucose.

The aim of our study was to evaluate the effect of low-power laser irradiation at two wavelengths (635 and 830 nm) on the secretion of inflammatory factors (TNF-α and IL-6) by the endothelial cell culture&#;HUVEC line (human umbilical vein endothelial cells)&#;under conditions of hyperglycemia.

Material and methods

Endothelial cells (HUVEC line) were derived from human umbilical veins by the enzyme method using collagenase according to the method described by Jaffe et al. [21]. Cells were cultured in M199 media supplemented with 20 % fetal bovine serum (FBS), 100 U/ml penicillin (Gibco® products), and growth factors 50 μg/ml endothelial cell growth supplement (ECGS&#;Corning Inc. USA) and heparin. ECGS was obtained from a bovine neural tissue. The cells were incubated at 37 °C in a humidified atmosphere with 5 % CO2. After 2&#;4 passages and seeding the cells in 6-well culture plates, the proper experiment was conducted. HUVECs were placed at a density of 7.5&#;×&#;104 cells per square centimeter.

With the exception of the control group, 30 mM/L glucose was added to the culture medium. The culture medium was changed every 3 days. The experiment was repeated three times with three independent cells isolations.

A semiconductor-based laser (Roithner Lasertechnik GmbH, Austria) was used to generate a visible laser beam with the wavelength of 635 nm (AlGaAlP) and the wavelength of 830 nm (GaAlAs) in the infrared. The authors have used the optoelectronic set for controlled, reproducible exposure of electromagnetic irradiation of biological structures in the spectral band of tissue transmission window 600&#; nm [22]. The power of laser sources was 30 mW for 635 nm and 60 mW for 830 nm. The power density at the cell-layer level measured by using a laser power meter (Gentec, Model SOLO2 R2, Canada) was 1.875 mW/cm2 for 635 nm and 3.75 mW/cm2 for 830 nm. The power was constant in all experiments. The distance between the laser source and the surface of application was 10 cm, the application was carried through an optical fiber, and there was 80 cm2 of irradiated area. The experiment was conducted in four groups: 1&#;no glucose in culture medium, no irradiation (control group); 2&#;glucose, no irradiation; 3&#;glucose, laser irradiation with wavelength of 635 nm, energy dose of 2 J/cm2; and 4&#;glucose, laser irradiation with wavelength of 830 nm, energy dose of 2 J/cm2. The time of laser irradiation was  s for 635 nm and 533 s for 830 nm. The cells were cultured for 7 days, with two irradiations on days 5 and 6. At the end of experiment, conditioned medium from each well of culture plates was collected and centrifuged for 10 min at ×g and frozen at &#;86 °C. After thawing, the concentration of TNF-α and IL-6 in the supernatant was measured by ELISA test (eBioscience, Vienna, Austria) according to the manufacturer&#;s instructions. The remaining cells on the bottom of each well were harvested by using trypsin and counted by Buerker hemocytometry. This method uses trypan blue dye according to the method described by Basso et al. [23]. The results of the concentration of the parameters in the supernatant from each well of culture plates were analyzed per number of cells in each well.

Statistical analysis was performed using Statistica 10.0 (StatSoft Inc.). The one-way ANOVA was used for parametrical analysis (proliferation), and Kruskal-Wallis test was used for nonparametrical comparisons (TNF-α and IL-6). Statistical significance was defined as P&#;<&#;0.05. The results were presented as mean (M)&#;±&#;standard deviation (SD) or median (Me), lower (Q1) and upper (Q3) quartile.

The approval of the Bioethics Commission of the NCU Collegium Medicum in Bydgoszcz was obtained, No KB/135/, date: 27 May .

Results

Figure shows the results concerning the effect of low-level laser therapy on TNF-α concentration in the supernatant from HUVEC culture which was grown in the high concentration of glucose in the culture medium (glucose concentration of 30 mM/L). The level of TNF-α in the control group (group 1) without glucose in the medium and without laser irradiation was 0.79 pg/105 cells. Its concentration was only slightly higher (0.85 pg/105 cells) in the unirradiated group 2 containing glucose in the medium. The cells from groups 3 and 4 were grown in medium with glucose and irradiated at the wavelength of 635 and 830 nm, respectively. The TNF-α level in group 3 was slightly lower and in group 4 notably lower when compared to the group 2 result. The concentration in group 4 was 0.55 pg/105 cells and was about 35 % lower than in group 2. However, the final outcome of the ANOVA testing was statistically non-significant and reported as 0..

Figure presents the concentration of interleukin 6 in the corresponding groups as in Fig.  . The concentration of IL-6 increased several times after the addition of glucose to the culture medium. There was a statistically significant difference between the three experimental groups (2, 3, 4) and the control group (P&#;=&#;0., P&#;=&#;0., P&#;=&#;0., respectively). No effect of the laser was observed. There was no increase of IL-6 concentration in groups 3 and 4 when compared to group 2. Difference between groups 2, 3, and 4 was statistically non-significant.

Figure shows the number of HUVECs which was the highest in the control group, while the lowest number was observed in group 2. This difference in relation to the control group was statistically significant (P&#;=&#;0.). The number of cells in group 3 was slightly higher compared to group 2 and in group 4 reached the level similar to the control group.

Discussion

High concentration of glucose present in diabetes causes damage to endothelial cells. It is accompanied by inflammation associated with the secretion of pro-inflammatory cytokines such as TNF-α and IL-6. An increased secretion of TNF-α and IL-6 under conditions of hyperglycemia is a finding observed by several authors [24&#;27]. It is accompanied by a reduction of cell proliferation [10, 11, 15]. However, in the study of Brandner et al. [28], level of TNF-α in cultured diabetic keratinocytes did not differ significantly from nondiabetic keratinocytes, although the level of TNF-α messenger RNA (mRNA) in the skin of patients with diabetes was significantly higher compared to those without diabetes. The authors explain this finding by the fact that keratinocytes are not a major producer of TNF-α in the skin. In our study, TNF-α was only slightly higher in cell cultures with the high glucose level in the medium compared to cells grown under normal conditions (Fig.  ). In contrast, the IL-6 level was significantly increased in the groups with glucose in the medium compared to the control group without glucose (P&#;=&#;0.; Fig.  ). IL-6 can inhibit the production of TNF-α [8, 29] which is why a considerable increase of IL-6 under hyperglycemic conditions might have an impact on the level of TNF-α. Other authors have also observed the significant increase of IL-6 concentration under conditions of elevated glucose level [24, 30, 31]. In our study, a much smaller number of endothelial cells cultured under hyperglycemic conditions compared to culture under normal conditions (P&#;=&#;0., Fig.  ) shows the negative impact of high glucose concentration on the proliferation and cell viability, as reported also by other authors [10, 12].

In hyperglycemia, glucose is subject to auto-oxidation process and non-enzymatic glycation which leads to the production of reactive oxygen species. An increased oxidation of the cofactor NADPH to NADP+ and the reduction of NAD+ to NADH are observed [32]. These reactions disrupt a balance between oxidants and antioxidants system leading to hypoxia and synthesis of advanced glication end-product (AGE). It is the effect of cytokines, mainly TNF-α [26]. Hyperglycemia increases the production of free radicals in the mitochondria, particularly superoxide anion. These processes exacerbate the oxidative stress [33&#;35] which damages cells and induces apoptosis [36, 37]. Apoptosis may be promoted also by DNA damage [11, 38] and irreversible changes in cytoskeletal organization at high glucose concentration [39, 40]. IL-6 secreted during the acute phase of inflammation has varied effects on cells in diabetes [30]. Pro-inflammatory or anti-inflammatory properties of IL-6 depend on the nature and function of cells and factors inducing inflammation. At high concentration, it can inhibit the production of TNF-α [41] which was confirmed in studies in mice [42]. The cited works have shown that the reduced level of IL-6 is associated with the impaired wound healing in diabetes.

Hyperglycemia induces the production of pro-inflammatory cytokines and growth factors by activating key signaling pathways associated with MAPK (mitogen-activated protein kinases), NF-κB (nuclear factor-κB), and STAT3 (signal transducers and activators of transcription) [27, 31, 43, 44] depending on ROS and oxidative stress. This is related to the effect of TNF-α on altering the redox reaction in the mitochondrial respiratory chain [35, 45, 46].

Although molecular mechanisms of low-power laser irradiation are still poorly understood, it is already known that the beneficial effects are associated with stimulation of cellular metabolism after energy absorption [47]. A stimulation of photoreactive proteins like cytochrome C occurs in the mitochondrial respiratory chain. This can increase the availability of ATP in the cells and have an influence on reactive oxygen species [48, 49]. An improvement of the cellular energy state and normalization of cellular functions is manifested by alleviating the pain and inducing the self-healing process [50]. Kwon [16] has found that laser irradiation of osteoblasts in a diabetic model at the wavelength of 635 nm reduces the level of generated ROS.

Studies in animals suggest beneficial effects of LLLT in case of hyperglycemia. LLLT (wavelength of 650 and 980 nm) in rats with induced diabetes has an antihyperglycemic effect without an impact on the blood morphology and biochemical profile [19]. Rabelo et al. [18] have also reported the beneficial effect of reduced inflammation in diabetic rats irradiated by HeNe laser at 632.8 nm.

The effects of LLLT on various types of cells depend on irradiation parameters such as dose and wavelength. Higher doses (10 or 16 J/cm2) cause reduction of cell viability and mitochondrial activity and increase the percentage of DNA damage [50, 51]. The wavelength is also important during irradiation of diabetic fibroblast cultures. The wavelength of  nm gave significantly worse results in wound healing than 632.8 and 830.0 nm [51, 52].

Our research confirms the beneficial effects of LLLT on cells cultured with high concentrations of glucose. TNF-α level in the group of cells cultured in medium containing high concentration of glucose decreased under the influence of laser irradiation when compared to the unirradiated group. This applies particularly to 830 nm (Fig.  ). P&#;=&#;0. is not far from significance. This result of testing in the analysis of variance could be significantly influenced by values in group 4 (glucose in the medium, LLLT λ&#;=&#;830 nm) with an average value of TNF-α lower about 35 % in comparison to group 2 (glucose in the medium, without irradiation). Many authors have also observed TNF-α reduction caused by laser irradiation [53, 54].

Our results show the significant increase of IL-6 induced by hyperglycemia (P&#;=&#;0.); LLLT did not cause significant changes in the concentration of this cytokine in the endothelial cell culture (Fig.  ). Some authors have reported a rise in the level of IL-6 induced by LLLT [30, 55, 56]. Houreld et al. [53] have observed only slight and statistically non-significant increase of IL-6. Some of the literature data have also reported decreased level of IL-6 under the influence of laser irradiation [56]. The result we have obtained may be in favor of beneficial effects of LLLT, since IL-6 reducing TNF-α level can therefore protect cells from the damaging effect of hyperglycemia. This issue is discussed and needs further research [8, 57].

Data from the literature suggest that LLLT promotes the metabolic activity of cells and stimulates their proliferation [15, 58], in case of cells grown under hyperglycemic condition as well [50, 52, 53]. These findings are consistent with our research applying in particular 830 nm (P&#;=&#;0.; Fig.  ). Hyperglycemic conditions significantly reduce the proliferation of HUVECs. The use of laser irradiation increases the proliferation of these cells to the level observed in the control group (non-irradiated cells in medium without glucose) (Fig.  ).

Conclusion

1. It appears that the adverse effects of hyperglycemia on vascular endothelial cells may be corrected by treatment with LLLT, especially at the wavelength of 830 nm. It causes the reduction of TNF-α concentration and enhancement of cell proliferation.

2. Due to the significance of endothelial cells in the pathogenesis of diabetes and a small amount of studies evaluating the impact of LLLT on these cells under conditions of hyperglycemia, further work on this subject is warranted.

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Protocol registration

This is a systematic review protocol for clinical trials. The aim of this systematic review is to investigate the safety and effectiveness of LLLT compared with active conventional treatment, sham LLLT, or no treatment, or as an additional treatment compared with active treatment alone, in patients with DPN. This protocol was registered with the International Prospective Register of Systematic Reviews on April (registration number: CRD; http://www.crd.york.ac.uk/prospero/).

Criteria for included studies

To be included in this analysis, studies will need to meet the following criteria regarding types of studies, participants, interventions, controls, and outcomes (Table 1).

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Types of studies

The randomized controlled trials (RCTs) that focus on LLLT in patients with DPN will be included. In contrast, reviews, case reports, animal experiments, meeting abstracts, and any publications without primary data or an explicit description of the methods will be excluded. Besides, we will only include the articles written in Chinese or English.

Participants

Adults (older than 18 years) with a clinically confirmed diagnosis of DPN will be included. The diagnostic criteria refer to the standards established by the Chinese Medical Association Diabetes Branch [22] or American Diabetes Association [23, 24]. We will exclude any RCTs that investigated patients with other types of peripheral neuropathy. Besides, patients with severe heart disease, liver and kidney dysfunction, mental illness, or malignant tumors will be not included.

Interventions

Currently, lasers with an output power of less than 500 mW are referred to as low-level lasers in the field of medicine. Common medical LLLTs include (but are not limited to) He-Ne lasers, semiconductor lasers, and CO2 lasers. RCTs will be included if they compare LLLT with any of the following control interventions: sham LLLT, no (specific) treatment except conventional diabetes treatments, or active conventional medical treatment. RCTs will also be included if they evaluate LLLT as an addition to another active treatment.

1. A.

   LLLT vs. sham LLLT

2. B.

   LLLT vs. no treatment

3. C.

   LLLT vs. active conventional medical treatment

4. D.

   LLLT plus conventional medical treatment X vs. conventional medical treatment X alone

Outcomes

The major outcomes for this review include NCV and clinical scores that assess neurological function and related symptoms.

NCV consists of motor NCV and sural sensory NCV. Clinical scores that assess neurological function and related symptoms include (but are not limited to) the Michigan Neuropathy Screening Instrument, Toronto Clinical Scoring System, Total Symptom Score, Neuropathy Deficit Score, and Neuropathy Symptom Score.

Outcomes will be recorded in two time periods: (1) short-term follow-up (less than or equal to 3 months and closest to 8 weeks after randomization) and (2) if available, long-term follow-up (more than 3 months and closest to 6 months after randomization).

Minor outcomes at both the short- and long-term follow-up will consist of quality of life such as the Short Form36 (SF-36), EuroQol five dimensions questionnaire (EQ-5D), and NeuroQol; pain assessment tools such as visual analog scale (VAS), neuropathic pain scale (NPS), Neuropathic Pain Questionnaire (NPQ), Short-form McGill Pain Questionnaire (SF-MPQ), and Brief Pain Inventory for Diabetic Peripheral Neuropathy (BPI-DPN); and adverse events associated with LLLT as a list of events.

Search strategy

Electronic searches

A systematic literature search will be conducted in the following electronic bibliographic databases: MEDLINE (PubMed), Embase, Cochrane Central Register of Controlled Trials, Web of Science (Science and Social Science Citation Index), Chinese National Knowledge Infrastructure (CNKI), VIP China Science and Technology Journal Database (VIP), WanFang Database, and SinoMed, from their inception to December . Two sets of keywords were chosen to identify the pertinent papers. The first set assessed the target population. The second set specified the type of intervention. A wildcard symbol (*) was used for generalizing keywords typically characterized by varying suffixes. The search was performed by inserting logical conjunctions (AND/OR) between the sets. Search areas included the &#;Mesh,&#; &#;title,&#; and &#;abstract&#; fields (Table 2). The articles need to be written in English or Chinese.

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Searching other resources

To overcome any deficiencies of the electronic databases, the following websites will also be searched for other clinical trial registries and gray literature about LLLT for DPN: Chinese Clinical Trial Registry (http://www.chictr.org.cn/), World Health Organization International Trials Registry Platform (www.who.int/trialsearch/Default.aspx), China Dissertations Database, GreyNet International (http://www.greynet.org), Grey Literature Report (http://www.Greylit.org/), Google Scholar (http://scholar.google.com/), Baidu Scholar (https://xueshu.baidu.com).

Study selection

All data retrieved through the performed search will be imported into Endnote X7, and duplicate data from the different databases will be removed. Two reviewers will independently screen the title and abstract of each study and make a decision to include it or not, according to pre-specified criteria. Next, the full text of the initially included literature will be retrieved. Two reviewers will carefully read the full text and make the final selections. Any discrepancies between the two reviewers will be resolved through consensus and by third-party adjudication, as needed. The study selection procedure is shown in Fig. 1.

Flow chart of the search process

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Data extraction

The following data will be extracted from each included study: first author name, publication year, sample sizes, design, age, sex, BMI, diabetes duration, fasting blood glucose (FBG), postprandial blood glucose (2hPBG), glycosylated hemoglobin (HbA1c), DPN duration, intervention descriptions, comparators, treatment duration, follow-up periods, outcomes, and adverse events (Tables 3 and 4). Two reviewers will independently extract the data, and any differences will be solved by discussion. We will contact the authors of any studies that do not report the aforementioned data (via ) to obtain the original data. We will conduct sensitivity analyses if the missing data are still not obtained.

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Risk of bias assessment

Two reviewers will independently assess the risk of bias of each study using the updated Cochrane Risk of Bias 2.0 tool [25]. This tool consists of six domains (randomization process, intended interventions, missing outcome data, measurement of the outcome, selection of the reported result, and overall bias). Each separate domain will be rated as having a low risk of bias, some concerns, or a high risk of bias. Disagreements between the two reviewers will be resolved through consensus and by third-party adjudication, as needed [26].

Strategy for data analysis

We will extract the main parameters of included articles according to the aim of this systematic review through Table 3, in which the article&#;s characteristics, methods, description of population, intervention descriptions, comparators, treatment duration, follow-up periods, outcomes, and adverse events will be included. After summarization of data, it will be determined if a meta-analysis is possible. If possible, Review Manager software version 5.3, provided by the Cochrane Collaboration, will be used to perform data synthesis and analysis. All experimental and control groups such as A, B, C, and D described in intervention will be evaluated as a single category.

Measures of treatment effect

We will select different evaluation methods according to the different types of data. Pooled dichotomous data will be presented as odds ratios or relative ratios with 95% confidence intervals. Pooled continuous data will be expressed as mean differences or standardized mean differences with 95% confidence intervals, depending on whether the measurement scale is consistent or not. Heterogeneity will be assessed by the chi-squared test and I2 statistic. If there is no heterogeneity (P > 0.1, I2 < 50%), the data will be synthesized using a fixed effect model. In contrast, a random-effects model will be used if (P < 0.1, I2 > 50%). Furthermore, we will use subgroup or sensitivity analyses to explore the potential reasons for the differences in heterogeneity. We will conduct a general descriptive analysis if a meta-analysis cannot be performed.

Assessment of heterogeneity

Statistical heterogeneity will be assessed by I2 statistic. When the I2 is greater than 50% (i.e., there is substantial heterogeneity), the possible sources of clinical heterogeneity are judged by combining the differences in population characteristics (e.g., comorbidity, age, gender, BMI, blood sugar indicators, DPN duration), intervention (e.g., different laser parameters such as energy density, wavelength, different intervention forms), outcome evaluation methods, and control selection in the studies included in the comparison of clinical knowledge, and then verified by subgroup analysis and sensitivity analysis.

Subgroup analysis

For the primary outcomes of NCV and clinical scores, the trials will be sub-grouped by dosage of LLLT such as energy density (greater than 3 J/cm2 or not), therapeutic schedules (whether or not sufficient treatment duration and sessions), BMI (&#; 24 kg/m2 or not), and blood sugar indicator of patients (FBG &#; 7 mmol/L and 2hPBG &#; 8 mmol/L, above this standard or no report)

Sensitivity analysis

In the sensitivity analysis, the following two types of studies will be excluded, one by one: (1) studies missing data, and (2) studies with a high risk-of-bias rating. The impacts of research quality, sample sizes, missing data, and statistical methods on the results of the meta-analysis will be evaluated by merging the data.

Reporting bias

If the number of studies is more than 10, a funnel plot will be constructed to examine publication bias.

Grading the quality of evidence

The quality of evidence for the entire study will be assessed using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach [27]. The limitations of study design, inconsistency, indirect evidence, inaccuracy, and publication bias will be evaluated. The GRADE system classifies the evidence into four levels: high, moderate, low, or very low.

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