سرطان زنان

The Effects of Synbiotic Supplementation on Serum Anti-Inflammatory Factors in the Survivors of Breast Cancer with Lymphedema following a

Low Calorie Diet: A Randomized, Double-Blind,

Clinical Trial

ABSTRACT

Background and Aim: Breast cancer-related lymphedema (BCRL) is a treatment-related inflammatory complication in breast cancer survivors (BCSs). This study was aimed to evaluate the effect of synbiotic supplementation on serum concentrations of IL-10, TGF-β, VEGF, adiponectin, and edema volume among overweight or obese BCSs with lymphedema

following a low-calorie diet (LCD).

Method: In a randomized double-blind, controlled clinical trial, 88 obese and overweight

BCSs women were randomized to synbiotic supplement (n = 44) or placebo (n = 44) groups and both groups followed an LCD for 10 weeks. Pre- and post-intervention comparisons were made regarding the anti-inflammatory markers which included IL-10, TGF-β, VEGF, adiponectin, edema volume, and anthropometric measurements. Also, the same factors were

analyzed to find inter-group disparities.

Results: There were no significant differences among participants in the baseline, except

for IL-10 and adiponectin. Post-intervention, no significant differences were observed regarding the anti-inflammatory markers, including IL-10, VEGF, adiponectin, and TGF-β between the groups. After 10 weeks of intervention edema volume significantly decreased in the synbiotic group; additionally, anthropometric measurements (body weight, BMI, body fat percent, and WC) decreased in both groups significantly (P < 0.001 and P < 0.005;

respectively).

Conclusion: Synbiotic supplementation coupled with an LCD in a 10-week intervention had

beneficial effects on increasing the serum TGF-β, IL-10, and adiponectin levels in women with BCRL. It also reduced arm lymphedema volume. Therefore, synbiotic supplementation can be effective in improving health status in BCRL patients.

Abbreviations: BCRL: Breast cancer related lymphedema; BCSs: Breast cancer survivors; LCD:

Low calorie diet; TNF-α: Tumor Necrosis Factor-α; BMI: Body Mass Index; IL-10: Interleukin-10;

TGF-β: Transforming growth factor-β; VEGF: Vascular endothelial growth factor; CFU: Colony

Forming Unit; FOS: Fructo-oligosacharide; CDT: Complete Decongestive Therapy

ARTICLE HISTORY received 9 May 2020 accepted 26 april 2021

Intoduction

Breast cancer is the most common cancer in women (prevalence rate of 24.2%) which leads to 15% of all cancer death among females worldwide (1). The most common causes of breast cancer are genetic and environmental factors (2). Due to progress in early diagnosis and effective use of treatments, the mortality rate of breast cancer has declined in recent years (3). Increased life expectancy of breast cancer survivors may cause higher frequency of treatment complications. Breast cancer-related lymphedema (BCRL) is one of those complications, which disrupts the performance and quality of life in affected patients (3). BCRL usually occurs after treatment of breast cancer with a prevalence rate of 24%–49% following mastectomy and 4%-28% following lumpectomy (4). Axillary lymph node dissection, radiation therapy, and obesity are the main risk factors for BCRL (5).

It has been suggested that obesity or being overweight is associated with the progression of lymphedema after breast cancer treatment (6–13). In contrast, weight loss effectively improves BCRL (14). Therefore, weight loss is recommended as a treatment option for obese patients with lymphedema (12).

Former studies propose inflammation as one of the possible pathways in the pathogenesis of lymphedema in breast cancer survivors (BCSs) (15).

Interleukin 10 (IL-10) is an anti-inflammatory cytokine (16) which induces immunosuppressive reactions by suppressing the secretion of inflammatory proteins such as interleukin 1-beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) (17). Transforming growth factor-beta (TGF-β) is another anti-inflammatory cytokine (18) which might prevent the onset of lymphedema potentially through its anti-inflammatory properties (19). Another hormone with possible anti-inflammatory effects is adiponectin which is secreted by the white adipose tissue (20). Adiponectin has been shown to improve lympho-vascular function, thus relieving lymphedema in a mouse model (21). Moreover, reduced levels of serum adiponectin have been shown to deteriorate lymphedema by reducing lymph-angiogenesis (21). Other factors worthy of note are the members of the vascular endothelial growth factor family (VEGF), including VEGF-C and VEGF-D; these factors contribute to angiogenesis and lymphangiogenesis, therefore ameliorating lymphedema through increased growth, proliferation, and migration of lymphoid endothelial cells (22, 23).

Probiotics play an immunoregulatory role by producing anti-inflammatory cytokines such as IL-10 and suppressing the production of inflammatory cytokines such as IL-1β (24). Probiotics are defined as “live microorganisms which when administered in adequate amounts confer a health benefit to the host” (25). Amdekar et al. showed that lactobacillus casei and lactobacillus acidophilus significantly reduce inflammation and edema induced by carrageenan in rats; they proposed that this effect might be due to their impact on decreasing the gene expression of inflammatory cytokines such as TNF-α and increasing the gene expression of IL-10 (26). Another pathway by which some probiotics may exert their anti-inflammatory properties might be their action in boosting the gene expression of transforming growth factor beta (TGF-β) (27). The results of a recent systematic review and meta-analysis also support the anti-inflammatory effects of some prebiotics (28).

Reduced levels of serum adiponectin have been shown to deteriorate lymphedema by reducing lymph-angiogenesis (26). Furthermore, it has also been observed in some experimental studies that probiotics can increase the concentration of serum adiponectin (29). It has also been shown that probiotic VSL # three might have a role in improving the angiogenesis and its attributed health effects via augmenting the gene expression and production of VEGF (30). VEGF induces angiogenesis and lymphangiogenesis and reduces lymphedema through increased growth, proliferation, and migration of lymphoid endothelial cells (22, 23).

Considering the current evidence regarding the possible effects of probiotics on improving the inflammatory response in various animal- and human-based investigations, we conducted the current study to examine the effect of synbiotic supplementation, coupled with a low-calorie diet (LCD), on serum consentration of IL-10, TGF-β, VEGF, and adiponectin; arm lymphedema volume; and also some anthropometric factors in overweight or obese BCSs with lymphedema.

We hypothesized that synbiotic supplementation increases the levels of anti-inflammatory markers among BCSs with lymphedema, reduces arm lymphedema volume, and also improves some anthrometric criteria including weight, body mass index, body fat percent, skin fold thickness, and waist circumference.

Methods

Study Design

This study was a 10-week, pre-post randomized double-blinded placebo-controlled clinical trial in BCSs with lymphedema, registered in the Iranian clinical trials website (http://www.irct.ir: IRCT2017092023861N6) and followed the Declaration of Helsinki and Good Clinical Practice guidelines. Study received ethical approval from Iran University of Medical Sciences ethics committee. The study was conducted from October 2017 to November 2018.

Participants

Women with arm lymphedema secondary to breast cancer were recruited from Seyed Khandan

Physiotherapy Clinic. Participants underwent conventional treatment for lymphedema, including the use of compression armsleeves and Complete Decongestive Therapy (CDT) that were monitored by a trained lymphedema therapist. Inclusion criteria included an age range of 18-65 years, having a body mass index (BMI) between 25 and 40 kgm−2, receiving no chemotherapy or radiotherapy during the previous 6 mo, and being in stage 1 or two of lymphedema. Exclusion criteria included taking multivitamin or omega-3 supplements up to one month prior to the study; enrolling in a structured weight loss program for at least 6 mo, prior to the study; having heart, kidney, liver, lung, and chronic or acute inflammatory disease; taking probiotic supplements for at least 6 mo, prior to their participation in the study; having serious food allergies; suffering from mental illnesses; suffering from endocrine diseases (such as thyroid abnormalities and diabetes), having gastrointestinal problems, smoking and alcohol consumption or lymphedema caused by disorders other than the one intended.

Randomization and Allocation

A total of 88 women with lymphedema were recruited into the study and were randomly assigned to receive either multispecies synbiotic supplements (n = 44) or the placebo(n = 44). All patients entered a 2-week run-in period during which they had to refrain from ingesting any other probiotic-enriched food. During the run-in period and at the end of the study, participants were asked to record their dietary intakes for 3 nonconsecutive days (two weekdays and a weekend day). Additionally, a low-calorie diet (LCD) was prescribed to all the participants at the beginning of the intervention which they were instructed to follow for the entire period of the study, lasting 10 weeks. The permuted block randomization with quadruple blocks was performed to randomize the participants. In order to randomize the study subjects (n = 88), 22 blocks were produced using an online site (www. sealedenvelope.com). To assure that the whole randomization process is absolutely double-blinded, unique codes, generated by the software, were used on pharmaceutical boxes with identical shape and color. By entering each individual into the study based on the sequence produced, the packet of the drug in which the code was registered was assigned to the individuals.

Weight Loss Program

At the end of the run-in period, individualized dietary regimens were tailored for and prescribed to all participants aiming at reducing the body weight to a standard, healthy body mass index.

Dietay regimens were designed to produce an energy deficit of 500-1000 kcal per day compared to the participants’ usual intake which had been already assessed using food record prior to the randomization. The minimum calorie intake prescribed was 1200 kcal. Calorie intake was calculated using the Mifflin-St Jeor formula. Macronutrient distribution in relation to calorie consumption was prescribed as: carbohydrates 55%-65%, fat 20%-35%, and protein 10%-15% of total calorie intake.

Intervention

The synbiotic supplements (Lactocare Synbiotics, kindly provided by Zist Takhmir Company, Iran) consisted of seven viable and freeze-dried strains, including 109 CFU/g of Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Bifidobacterium breve, Bifidobacterium longum, Streptococcus thermophiles, and 38.5 mg of fructo-oligosaccharides (FOS). Placebo consisted of starch, lactose, magnesium stearate, and silicon dioxide (Zist-takhmir Co., Tehran, Iran) which was similar to the synbiotic supplements in color, shape, size, taste, and packaging. Study subjects were randomly assigned to receive either the placebo or multispecies synbiotic supplements for 10 weeks. All participants were asked to take one supplement daily. The patients were asked not to take more than one synbiotic or placebo capsule a day. They were also asked to avoid consuming any other probiotic foods, such as probiotic dairy and fermented products.

During the study, dietary advices and interventions were given by the same dietitian. Placebo or multispecies synbiotic supplements were provided for participants monthly. Compliance with consumption of capsules was monitored once a week through phone interviews.

The placebo and probiotic supplements were packaged in identical sealed boxes, identified by a code number only. Patients were instructed to keep the study medications refrigerated (between 2 and 7˚ C) throughout the study.

Assessment of Variables Anthropometric Assessments

Anthropometric criteria were assessed at baseline and after 10 weeks of intervention. Body weight was measured in an overnight fasting status, without shoes and in minimal clothing, using a digital scale (Seca, Hamburg, Germany) to the nearest 0.1 kg. Height was measured using a nonstretched tape measure (Seca) to the nearest 0.1 cm. BMI was calculated as weight in kilograms divided by height in meters powered by two. Waist circumference was measured to the nearest 0.5 cm at the end of normal expiration, at midpoint between the top of the iliac crest and the lower margin of the last palpable rib in midaxillary line.

Furtheuremore, skinfold thickness was determined at 4 sites (triceps, biceps, subscapular, and suprailiac) using Harpenden skinfold calipers (31). Measurements of skinfold thickness were conducted on the unaffected arm by the same investigator who was trained and practiced in the technique.

Dietary and Physical Activity Assessments

The dietary intake of participants at the baseline and post-intervention was assessed using food diaries (two weekdays and a weekend days); the obtained data were then registered and analyzed using Nutritionist IV software (First Databank, San Bruno, Calif, USA) modified for Iranian foods. Moreover, physical activity of each participant was assessed using the International Physical Activity Questionnaire (IPAQ) monitor at baseline and at the end of study.

Edema messurment

The water displacement method was used to obtain the volume of the edema. The healthy limb and then the affected limb were submerged in a water tank up to 2 cm below the armpit; edema volume was calculated as the volume difference between affected and unaffected arms in milliliters (32). All measurements were conducted by an experienced examiner who was unaware of the study-group assignments.

Biochemical Assessment

Ten milliliters blood samples were obtained from antecubital vein at the baseline and post-intervention, both after 12-hour fasting, and were immediately centrifuged (Hettich D-78532, Tuttlingen, Germany) at 3500 rpm for 10 mins, to separate the serum; the serum samples were then stored at −80 °C until being analyzed at the laboratory. Serum concentrations of IL-10, TGF-β, VEGF, and adiponectin were assessed by the use of ELISA kit (eBioscience, US) which took advantage of the double-antibody sandwich technique. The lower limit of detection for IL-10,

TGF-β, VEGF, and adiponectin were defined as

2.48 pg/ml, 5.11 ng/l, 10.42 ng/l, and 0.11 mg/l; respectively. All clinical and laboratory data were collected in a double-blind manner.

Sample Size

To estimate the acceptable sample size, the serum IL-10 level was considered as the primary outcome in this study. Secondary study outcomes included TGF-β, VEGF, and adiponectin. Based on the amount of Cohen’s standard effect, we considered a type I error of 5% (α = 0.05), type II error of 20% (β = 0.2; power = 80%), and the Cohen standardized effect value of 0.65 (one effect at moderate level). Considering the serum IL-10 level as the key variable, a sample size of 44 patients for each group was then calculated.

Statistical Analysis

To ensure a normal distribution of variables, histogram and Shapiro-Wilk tests were applied. The intention-to-treat (ITT) analysis was applied for all randomly allocated subjects. In order to describe the quantitative variables, the mean (±standard deviation) or the median (first quartile and third quartile) and to demonstrate the qualitative variables, frequency report (as percent) were used.

To compare the mean of quantitative outcomes between the two groups, independent-samples t-test (for age, age at cancer diagnosis, dietary intakes, weight, BMI, fat percent, TSF, WC, and PA) or its nonparametric counterpart, Mann-Whitney test (for time past treatment of cancer, duration of lymphedema, tumor size, range of motion, CDT course numbers, removed lymph nodes, affected lymph nodes, edema volume, serum concentrations of IL-10, TGF-β, VEGF, and adiponectin) were used. We also took advantage of the paired t test or its nonparametric counterpart, the wilcoxon test, to identify within-group differences (before and after intervention). Chi-square test or Fisher’s exact test was used to compare qualitative factors (including lymphedema stage, surgery type, chemotherapy, radiotherapy, breast cancer stage at diagnosis, estrogen receptor, progesterone receptor, HER2, current endocrine therapy, herceptin injection, homogeneity of hands, fibrotic tissue stages, occupation, marital status, and education) between the two groups.

Delta (Δ) values were used to verify the differences in the variables before and after the intervention. We used ANCOVA test to compare post-intervention outcomes between the 2 groups by adjusting the baseline values of each outcome to the baseline BMI as covariate. Spearman correlation analysis was performed to evaluate the relationship between changes in body weight/waist circumference and the blood factors/edema volume differences. P-value <0.05 was considered as statistically significant. Analyses were conducted using statistical software, SPSS version 24 (SPSS Inc., Chicago, IL). Non-parametric ANCOVA analysis was performed with R software (package “sm” in R-3.5.1 for windows).

Participants

A total of 352 women were primarily recruited to participate in the study out of which 88 women who met the inclusion criteria were permitted to enroll in the study and were randomly assigned to two groups. The flow chart of study

Age distribution of participants was 53.80 (± 9.16) years in synbiotic group and 52 (± 7.95) in placebo group at baseline. As shown in Table 1, there were no significant differences in demographic characteristics between the two groups at the baseline (Table 1). No significant differences were observed in variables related to the lymphedema, including age at diagnosis of breast cancer (year), time since diagnosis of lymphedema (month), time since end of breast cancer treatment (year) and so forth at the baseline of the study (Table 1).

Dietary Intake Outcomes

Dietary intake of energy, macro- and micro-nutrients obtained from 3-day dietary food records, were not significantly different in 2 groups at baseline and after 10 weeks of intervention (Table 2).

However, dietary consumption of energy, carbohydrate, protein, total fat, SFA, PUFA, MUFA, cholesterol, sodium, phosphorus, selenium, and beta-carotene decreased significantly in two groups following 10 weeks of intervention (P < 0.05).

Anthropometric Measurements and Physical Activity Level

As depicted in Table 3, weight (kg), BMI (kg/m2), body fat percent (%), skin fold thickness (TSF) (cm), waist circumference (WC) (cm) and physical activity (PA) (min/week) were not significantly different between the two groups at baseline and after 10 weeks of intervention. However, within-group comparisons indicated that weight, BMI, body fat percent, TSF and WC, were significantly reduced in both groups (-2.48 ± 3.03 kg, −1.73 ± 2.26 kg for weight, −0.95 ± 1.14 kg/m2, −0.68 ± 0.90 kg/m2 for BMI, %-1.14 ± 1.37, %-0.83 ± 1.09 for fat percent, 0 (-3.0, 0), 0 (-2.0, 0) cm for TSF and −3.89 ± 3.26 cm, −3.84 ± 3.60 cm for WC in synbiotic and placebo group, respectively; P < 0.005) compared to the baseline values.

Physical activity levels increased significantly in both groups over 10 weeks of intervention in the symbiotic group [17.0 (3.25, 30.50) min/week] and the placebo group [21.0 (7.0, 33.0) min/week] (Table 3).

Post-intervention, edema volume significantly decreased only in the synbiotic group compared to the baseline values (-149.26 ± 205.91 mm3, P < 0.001) (Table 3).

Anti-Inflammatory Markers

Due to the necessity of using multiple testing models to analyze the secondary outcomes, we used Bonferroni correction test to calculate between- and within-group cahnges in VEGF, adiponectin, and TGF-β levels. The corrected significant level was then defined as 0.016. At the baseline and post-intervention, anti-inflammatory markers

including IL-10, VEGF, adiponectin, and TGF-β concentrations were not significantly different between the two groups, except for IL-10 and adiponectin at the baseline (Table 4). IL-10 levels significantly decreased in the placebo group (-48.48% (16.5– 10.00); P= < 0.001) while showing no significant changes in the synbiotic group. On the other hand, synbiotic supplementation significantly prevented the reduction of IL-10 levelscompared with the placebo group (adjusted P = 0.004). Also, the synbiotic supplementation caused a significant reduction in VEGF levels (P = 0.001). Within-group analyses revealed a marked increase in adiponectin levels only in the synbiotic group (51.12% (1.33–2.01); P < 0.001). Using Bonferroni test adjusted for baseline measurements, we observed that synbiotic supplementation did not exert any significant impact upon adiponectin levels compared with the placebo (P = 0.444). Such lack of statistically marked improvement was also observed regarding the TGF-β levels in the placebo group P = . Compared with placebo, synbiotic supplementation significantly boosted the TGF-β levels

Using the Pearson correlation coefficient test, we were not able to detect any statistically significant correlations between anti-inflammatory markers and body weight or WC (Table 5). However, edema volume changes significantly correlated with body weight (r = 0.326, P = 0.038) and WC (r = 0.330, P = 0.035).

Discussion

To the best of our knowledge, this research is the first study conducted to investigate the effects of synbiotic supplementation on the anti-inflammatory markers, anthropometric parameters, and arm lymphedema volume among overweight and obese patients with

BCRL. Our study revealed that consumption of multispecies synbiotic supplements, compared with the placebo, for 10 weeks, is able to prevent the reduction of IL-10 and VEGF levels and reduce arm edema volume in BCRL patients. Even though the consumption of synbiotic supplement led to higher serum concentrations of TGF-β and adiponectin, but these values were not significantly different between the two groups after 10 weeks of intervention.

The significant improvement in anthropometric indices, including body weight, BMI, fat percentage, and waist circumference in both groups at the end of the intervention could be attributed to receiving a low-calorie diet by all patients. Consumption of supplemental synbiotic did not create any further improvements in these measurements when compared to the placebo.

Synbiotic Supplementation and Arm Edema Reduction in excess arm edema volume after synbiotics supplementation is a new finding that has not been reported prior the present study. Reduction of arm edema volume occurred in both groups, but this reduction was only significant in the synbiotic group (%- 37.26 vs %- 20.75). However, in previous studies, the effect of weight loss has been shown to improve edema (14, 33, 34). The results of the present study unprecedentedly prove that receiving synbiotic supplement along with a weight-loss diet can significantly reduce the arm’s edema. Shaw et al. showed thata weight-loss diet lasting 12 weeks could significantly reduce the volume of edema (33). Obesity is a risk factor for the development and progression of lymphedema in BCSs (6, 12). Several mechanisms are proposed for this relationship, including a reduced muscle pump efficiency, excess fat deposits, and negative effects on lymphatic vessels in overweight or obese patients (35, 36).

On the other hand the fermentation of polysaccharides in the intestine increases the transfer of carbohydrates to the liver and adipocytes and activates the lipogenic enzymes, such as acetyl-CoA carboxylase. These changes eventually lead to the accumulation and storage of triglycerides in the liver and adipose tissue. Probiotics decrease the intestinal polysaccharide fermentation by altering the intestinal microflora and has an inhibitory effect on this cycle (37, 38). The intestinal microbiota also produces conjugated linoleic acid (CLA) which could induce the apoptosis of adipocytes and subsequently weight loss, possibly by inhibiting nitric oxide synthesis, reducing calorie intake, and increasing energy expenditure (39, 40).

Synbiotic Supplementation and Anthropometric Measurements

According to our findings, the prescribed low-calorie diet resulted in weight loss in all patients, but this reduction was higher in the synbiotic group (%- 3.11 vs %- 2.21). However, this difference was not statistically significant. In a meta-analysis study, the final results revealed that the consumption of probiotics compared to placebo significantly reduces weight, body mass index, and fat percentage; in contrast, we were not able to show the same kind of causal relationship (41). This lack of effect might be in part due the synergistic effects of cancer/chemotherapy and obesity on the inflammaory status of the body which in turn may have overruled the existing impact of synbiotic supplementation on weight loss. One possible mechanism to describe the impact of synbiotic supplementation on reducing the edema in these patients could be their effect on improving the inflammatory status of the body; this postulation is further explained in the next section.

Synbiotic Supplementation and AntiInflammatory Factors

The present study showed that taking synbiotic for 10 weeksis able to prevent the reduction of IL-10 and VEGF levels in BCRL patients compared to the placebo group. However, after these figures were adjusted according to the baseline characteristics of the patients, the significance of between-group differences disappeared, with regard to adiponectin and TGF-β levels .

In line with our study, in a meta-analysis of six clinical trials with the mean intervention period of 8 week in healthy subjects, probiotic consumption did not exert any significant effects on TGF-β levels (42). However, some studies conducted on patients with atopic dermatitis and Parkinson’s (43–45) were able to detect some of the impact that synbiotic supplementation may have on TGF-β levels. It is worthy of note, though, that these diseases have some autoimmune responses as part of their underlying pathogenesis (46, 47). Based on these two contrasting findings, we could postulate that the existence of an already modified immune response, might magnify the possible impact of synbiotic or probiotic supplementation on TGF-β levels in different populations.

We also observed that IL-10 levels did not change in the synbiotic group, while were significantly reduced in the placebo group. Also, after statistical adjustment to the baseline characteristics, synbiotic supplementation significantly prevented a further reduction in interleukin-10 levels compared with the control gropu. Lohela et al reported that Lactobacillus casei supplementation for 8 weeks in rheumatoid arthritis patients resulted in a significant increase of IL-10 levels (48). However, a meta-analysis study concluded that supplementation with synbiotic and probiotic in healthy individuals creates no further benefits regarding serum IL-10 (42). This contrast might be best justified by the presumption that low-level inflammation could alter the effectiveness of synbiotic or probiotic supplementaion. In other words, we can draw such deduction to say that anti-inflammatory effects of synbiotic supplementation does in part depend on the hosts’ inflammatory status; based on the fact that our study subjects (BCSs) also manifest a low-level inflammation.

Dysbiosis is a condition in which gastrointestinal microbial homeostasis is disrupted and often leads to health problems. One of the causes of dysbiosis is obesity (49, 50). Dysbiosis, in turn, may induce inflammation; lipopolysaccharides (LPS), also called endotoxins, derived from the outer cell walls of the gram-negative bacteria, bind to the Toll-like receptor 4 (TLR4) which subsequently induces NF-κB in the intestinal epithelial cell.The NF-κB transcription factor causes systemic inflammation by reducing the production of anti-inflammatory cytokines, such as IL-10 (51–53). Probiotics increase the secretion of anti-inflammatory cytokines, such as IL-10, by altering the differentiation and the activity of immune cells, including dendritic cells (54). IL-10 expression in T cells is regulated by TGF-β (55).

We also observed a significant reduction an serum VEGF levels only in the control group. Additionally, serum VEGF levels were significantly different between the two groups at the end of the intervention, after being adjusted to the baseline characteristics.

Although several factors contribute to the angiogenesis cascade, VEGF is the main angiogenic factor (56). In line with our findings, It has been shown that

Lactobacillus rhamnosus can improve gastric ulcer in rats by increasing the expression of VEGF (57). Marliks et al reported that although probiotic supplementation did not improve VEGF levels within each group of intervention or placebo, there was a significant difference between the two group at the end of the intervention in patients with cirrhosis. This finding matches our results on the effect of synbitic supplementation on VEGF serum levels (58). Some studies have suggested that IL-10 has an angiogenic effect via its effect on VEGF (56, 57). Based on this hypothesis, the VEGF-boosting effect of synbiotic supplementation is attributable to its impact on increasing IL-10 levels.

Regarding another important inflammatory factor, we were also able to detect a significant improvement in adiponectin levels after receiving the synbiotic supplement. Nonetheless, the two groups were not significantly different at the end of the study. Similar observations were made in previous studies (59, 60). Even though Feizollahzadeh et al showed that the milk enriched with Lactobacillus plantarum (61) could not improve serum levels of adiponectin, an experimental study has shown that the serum concentration of adiponectin increases after receiving probiotics. Also in contrary to our findings, in another study on obese indiviuals, consumption of fermented milk caused a significant increase in blood adiponectin levels (62). Circulating adiponectin concentration increases during weight loss in animals and humans (63). Although there was a significant weight loss in both groups after the period of intervention, we were unable to detect any between-group differences; a finding that might explain the lack of post-intervention between-group differences in adiponectin levels.

Limitations of Study

One limitation of this study is the lack of a follow-up assessments to observe the lasting effects of the intervention; therefore, the findings are only valid for immediately after the completion of the study. Another limitation is the age dependency of the effect which was not investigated. The mean age of participants was 53 years; thus, we can not extrapolate the findings to younger or older survivors. Lack of fecal bacterial load measurements and microbiome characterization at baseline, during, and after the synbiotic supplementation was another limitation in our study.

Suggestions for Future Research

We propose that future studies be conducted to further deepen our understanding on the imapct of synbiotic supplementation on inflammation at the gene-expression level. Considering their confounding or causal effect in future studies, design may be valuable . Generalizing the results of present study to men or other types of cancer survivors should be done with caution. Confirmation of our results requires more interventional studies in this field. Furthermore, specific studies are needed for understanding the mechanisms involved between adiposytokine levels and edema volume in the breast cancer survivors.

Conclusions

Synbiotic supplementation, lasting for10 weeks, in women with BCRL had beneficial effects on VEGF and IL-10 levels. It also reduced arm lymphedema volume. Therefore, synbiotic supplementation can be effective in improving health status in BCRL patients.

Acknowledgments

We would like to gratefully thank the participants for their support in the study. We are also thankful to Seyed-khandan rehabilitation Center and Zist Takhmir Company for their assistance in this study.

Conflict of Interest Statement

The authors report no conflict of interest.

Author Contributions

MZ, AST, and SHH provided study concepts and designed the study. AST, SHH, HE, ZS, and MI helped in data acquisition. MZ checked quality control of data and algorithms. LJ, MZ, AST, SV, and MN took part in data analysis, interpretation, and statistical analysis. AST, MZ, ShJ and HE contributed to manuscript preparation, editing, and review.

Funding

This study was supported by Iran University of Medical Sciences (IUMS) with 96-02-27-31431 grant number