The religious form of IF (i.e.: Ramadan model) was shown to mitigate low-grade systemic inflammation, reduce pro-inflammatory cytokines and OS markers, and confer a short-term transient cardiometabolic protection [11, 15, 33].
While more than one study examined the effect of observing IF regimens on genetic expression (e.g., FTO, Nrf2, TFAM, SOD2, SIRT1, SIRT3, and CLOCK)[34–39], few studies have examined the impact of genetic variations on the metabolic response to IF regimen among overweight and obese subjects. In these, the IF was associated with higher weight reduction in comparison to low-calorie diet among overweight/obese people with GG genotype of the UCP2 gene, while no differences in weight loss were found between the two regimes among people with AA + GA genotype . To our knowledge, this is the first study investigating the impact of Hp phenotypes (Hp1-1, Hp2-1, and Hp2-2) in obesogenic (including bodyweight reduction), metabolic and inflammatory markers in response to IF regimen. Our results demonstrated that the Hp phenotype might independently influence the outcomes of IF on abdominal obesity, lipid profile, inflammatory cytokines, serum Hp, and CD163 in overweight and obese individuals. Our results confirmed the momentous role of IF in reducing low-grade systemic inflammation and enhancing anti-inflammatory mechanisms, thereby mitigating health deteriorations accompanying people with obesity.
Impact of RIF on serum Hp, CD163, and other measured parameters
The effect of IF and caloric restriction on body weight change has been the subject of several studies; numerous reviews and original research have been published in attempts to elaborate on this perpetuate effect [3, 6, 41, 42]. Given that fasting during Ramadan represents a form of IF and TRF  that is globally observed by at least 1.5 billion Muslim people each year, the impact of RIF on body weight loss and the associated metabolic and inflammatory effects needs further elaboration.
The present study suggests that RIF is associated with reduced body weight, body fat percentage, and visceral fat area, with improvements in several cardiometabolic risk factors. These findings concerning body weight and composition are consistent with those of a previous study that reported significant reductions in body weight, fat mass, BMI, visceral fat area, and fat-free mass in healthy adults following Ramadan fasting during the summer [44, 45].
Additionally, IL-6 and TNF-α levels had significantly decreased, whereas IL-10 levels had significantly increased at the end of the Ramadan month. These significant reductions in TNF-α and IL-6 after observing RIF were consistent with significant reductions reported in a similar previous study on the Ramadan model of IF . Furthermore and as discussed above, the secretion of pro-inflammatory cytokines IL-6, TNF-α, and anti-inflammatory cytokine IL-10 may be regulated by the Hp phenotype . Besides, pro-and anti-inflammatory cytokines play crucial roles in the upregulation and downregulation of CD163 expression, respectively . Hp expression depends on the Hp phenotype and the levels of the pro-inflammatory cytokines IL-6 and TNF-α, both of which have a crucial role in Hp concentration . Therefore, Hp phenotype and Hp-Hb complex have leading roles in the regulation of serum Hp and CD163 levels. Several studies have shown that the pro-inflammation characteristics of serum Hp [30, 49], and the anti-inflammation activity of CD163  altered in response to different Hp polymorphisms. These findings were consistent with our previous study that showed RIF had an ameliorating effect on pro-inflammatory cytokines and in enhancing the anti-inflammatory response [12, 45, 51]. The present results confirmed a significant increase in the anti-inflammatory serum CD163 and a significant decrease in the pro-inflammatory serum Hp following RIF when compared with pre-fasting levels. There may be, however, other unexamined factors that have an impact on these variables during the Ramadan month, such as changes in circadian rhythm that may influence IL-6 production, as reported in non-fasting research .
It is well established that the increased ratios of anti-inflammatory (IL-10) to pro-inflammatory (TNF-α and IL-6) cytokines represent a protective factor against atherogenesis . In one study , the ratios of IL-10 to TNF-α and IL-10 to IL-6 showed preferred increments at the end of the fasting month. Their reported noticeable reduction in the IL-6:IL-10 ratio at the end of Ramadan suggests that RIF had a favorable protective effect against systemic inflammation and subsequent metabolic derangements .
Therefore, the lower level of IL-6 at the end of RIF in the present study could be explained by the decreased level of physical activity reported by most subjects during the month of Ramadan and in the literature . A recent study showed that RIF is associated with reduced activity and sleeping time without changes to the resting metabolic rate or total energy expenditure . This is consistent with the 0.5% reduction in muscle mass reported at the end of Ramadan.
HDL level was significantly increased at the end of RIF in this study. This finding was consistent with a previous study involving 81 fasting subjects, in which HDL decreased significantly at the end of RIF . This result was inconsistent with the findings of the majority of previous studies on RIF, where HDL increased or remained unchanged during the Ramadan month . A systematic review and meta-analysis of 57 studies investigating RIF among healthy subjects (N = 2771) showed a significant, but small, pooled reduction in HDL at the end of Ramadan fasting .
This discrepancy in the effect of RIF on lipid profile and HDL may be attributable to differences in cultural foods and dietary practices among different populations, especially in the types and amounts of dietary fats consumed. Further, the genetic makeup of study subjects affects the way their bodies respond to various changes during the Ramadan month, which in turn affects their lipid profile at the end of the fasting month.
It has been proposed that the fasting state (i.e.: Ramadan IF) induces an elevation in free fatty acids and ketone bodies, such as beta-hydroxybutyrate, which in turn may impose damage on the metabolically active mitochondria in the neurons of the fasting organism. This damage, however, can be corrected by several mechanisms, including upregulated antioxidant defense genes and enhanced mtDNA repair .
The interplay between Hp polymorphism and IF in relation to CD163 and other measured parameters
The present study found that obese individuals with the Hp1-1 phenotype experienced a higher decrease in BW and BMI after RIF than those with the Hp2-1 or Hp2-2 phenotypes, with the differences in the change of BW being significantly different among the three Hp phenotypes. Overall, each Hp polymorphism underwent a significant decrease in BW and BMI due to RIF. Although expected, this result was interesting as there were significant changes in body weight and BMI in response to RIF in general. In addition, these results support the hypothesis of a previous study that suggested that the phenotype of Hp has a crucial role in modulating the oxidative-antioxidative status in both obesity and diabetes . Furthermore, our results showed a significant reduction in body fat percentage, fat mass, fat-free mass, and muscle mass for all of the three Hp phenotypes. Phenotype Hp1-1 had a tendency also to lose the greatest muscle/fat-free mass in comparison to the other two phenotypes, with the difference being significantly different, particularly against the Hp2-1 phenotype.
Previous studies showed that BMI is an indicator of total body fat, whereas waist circumference reflected visceral fat. Visceral fat deposition is known to have more metabolically adverse effects than subcutaneous fat . Therefore, waist circumference measurement is a better indicator of metabolic and inflammatory disorders related to obesity. Both waist circumference and BMI showed the strongest positive correlations with Hp among the various obesity measures [62, 63]. In a recent study, waist circumference and visceral fat area were found to significantly decrease in Hp2-1 after RIF compared with before RIF, whereas Hp1-1 and Hp2-1 showed non-significant reductions.
Interestingly, the WC reduction experienced by obese individuals withHp1-1 phenotype following RIF compared was significantly different from the ones of Hp2-1 and Hp2-2. Similarly, a recently published paper that investigated hypo-caloric dietary programs among obese women showed these dietary programs had a stronger positive influence on abdominal obesity (waist circumference, total body fat, and fat mass) of the Hp1-1 phenotype compared with the Hp2-1 and Hp2-2 phenotypes . That study supported our finding that individuals with an Hp1-1 phenotype had higher expression of anti-inflammatory cytokines and were more resistant to OS than Hp2-1 and Hp2-2 individuals. Incidentally, Hp2-1 and Hp2-2 individuals have been reported to have a higher incidence of risk factors for obesity, diabetes, and cardiovascular diseases .
Due to its association with pro-inflammatory activity, higher levels of Hp have been associated with an increased risk for obesity, type 2 diabetes, and cardiovascular diseases [65–67]. Many studies have suggested that Hp polymorphism not only affects serum Hp but also influences lipid profile levels. In other words, there is a positive correlation between serum Hp and TC, non-HDL-C, HDL-C, and TG concentrations in obese individuals . Our results are consistent with this finding. Moreover, several studies [67, 69], including the present study, demonstrated a higher concentration and positive association of LDL-C in obese Hp2-2 individuals compared with obese Hp1-1 individuals. Furthermore, the present study showed a significant decrease in TG and LDL in both Hp2-1 and Hp2-2 subjects after RIF. Additionally, HDL-C levels were significantly higher following RIF in all Hp polymorphisms. Conversely, TC was significantly reduced in all of the Hp phenotypes. The current results suggest that Hp2-2 individuals with obesity have more anti-inflammatory characteristics and will have a greater response to RIF and obtain optimum benefits from IF than those with Hp2-1 and Hp2-2 phenotypes.
The present study investigated the role of the environmental factors in modulating anti-inflammatory markers (IL-10) and pro-inflammatory cytokines (IL-6 and TNF-α) in response to IF in people with different Hp polymorphism phenotypes. It also explored the expression of an essential anti-inflammatory factor, CD163, in both RIF and Hp polymorphism. With the observed interesting effect of RIF on significantly reducing levels of IL-6 and TNF-a in all Hp phenotypes, this confirms previous findings demonstrating the positive impact of the Ramadan model of IF on the health of obese individuals . In contrast, IL-10 showed a significant increase in all of the Hp phenotypes after observing RIF.
As previously noted, serum Hp and CD163 levels are associated with and regulated by the pro-and anti-inflammatory cytokines (IL-6, TNF-α, and IL-10) alongside Hp polymorphism . There was a significant increase in serum of the anti-inflammatory CD163 and a considerable decrease of pro-inflammatory serum Hp in response to RIF. Additionally, serum CD163 levels were significantly increased in all of the Hp phenotypes after RIF, particularly Hp2-2 whose increase was significantly different from the Hp2-1 and Hp2-1 groups. Moreover, the serum CD163 mean difference values between Hp phenotypes revealed a trend in increment through Hp1-1, Hp2-1, and Hp2-2 respectively, but the changes were not significant between Hp1-1 and Hp2-2. In contrast, serum Hp showed significantly lower expression in the Hp2-1 and Hp2-2 phenotypes, yet an increase was experienced by the Hp1-1 phenotype after RIF.
Understandably, the Hp2-2 phenotype is mainly associated with increased risks for obesity, diabetes, and cardiovascular diseases, in addition to enhancing pro-inflammatory metabolites. RIF also has a positive impact on modulating and strengthening inflammatory cytokines. Interestingly, our findings revealed that Hp2-2 had a better response in moderating and enhancing anti-inflammatory markers than Hp1-1, which highlighted the influence of RIF on health outcomes for people with obesity, suggesting metabolites may offer a pathway to a healthier positive response.
Our results confirmed the decisive role of IF in reducing low-grade systemic inflammation and OS and enhancing anti-inflammatory mechanisms, thereby improving the health conditions of individuals with obesity. Moreover, this provided an example of the epigenetic factors of the different Hp phenotypes in response to RIF and confirmed that Hp1-1 individuals had a higher anti-inflammatory response than Hp2-1 and Hp2-2 individuals. Because of the positive impact of IF on the health of individuals with obesity in developing and enhancing adipocytokine pathways, Hp2-2 expressed a higher anti-inflammatory response to RIF compared with Hp1-1. Therefore, we propose that IF contributes to health benefits for individuals with obesity in moderating and refining their oxidative and inflammatory mechanisms.
In conclusion, four consecutive weeks of dawn-to-sunset IF was found to significantly and variably affect the anthropometric, metabolic, and inflammatory markers in relation to Hp polymorphisms. Our results confirmed the decisive role of IF in reducing low-grade systemic inflammation and OS and enhancing anti-inflammatory mechanisms, thereby improving the health conditions of individuals with obesity. Moreover, this provides an example of the epigenetic factors of the different Hp phenotypes in response to IF and confirmed that Hp1-1 individuals had a higher anti-inflammatory response than Hp2-1 and Hp2-2 individuals.