Mediterranean Diet and NAFLD: What We Know and Questions That Still Need to Be Answered

Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome and is expected to become the leading cause of end-stage liver disease worldwide over the next few decades. In fact, NAFLD encompasses different clinical scenarios, from the simple accumulation of fat (steatosis) to steatohepatitis (NASH), NASH-cirrhosis, and cirrhosis complications. In this context, it is fundamental to pursue strategies aimed at both preventing the disease and reducing the progression of liver fibrosis once liver damage is already initiated. As of today, no pharmacological treatment has been approved for NAFLD/NASH, and the only recommended treatment of proven efficacy are life-style modifications, including diet and physical exercise pointing at weight loss of 5%–7%. Different dietetic approaches have been proposed in this setting, and in this review, we will discuss the evidence regarding the efficacy of the Mediterranean Diet as a treatment for NAFLD. In particular, we will report the effects on liver-related outcomes.


Introduction
NAFLD is considered the hepatic manifestation of metabolic syndrome (MetS) and is defined as the accumulation of fat in the liver in patients who do not consume excessive amounts of alcohol (i.e., more than 20 g/day for women and less than 30 g/day for men). NAFLD encompasses different clinical scenarios, from the simple accumulation of fat in the liver (steatosis), to steatohepatitis (NASH), cirrhosis, and its complications [1].
The burden of disease related to NAFLD is extremely high in the general population, and the global prevalence of liver steatosis is indeed around 25%, reaching a pike of 46% in the United States [2]. These percentages have gradually increased in the past decades, parallel to rising rates of obesity and diabetes, to which NAFLD is tightly related. It needs to be pointed out that, owing to the increasing prevalence of the disease and to the prolonged life-expectancy, NASH-related cirrhosis is intake. As far as alcohol use is concerned, a moderate intake of ethanol is an additional characteristic of the MD (in particular, wine) [24]. A synthetic example of the recommended composition of a meal in the MD is given in Figure 2.
Nutrients 2019, 11, x FOR PEER REVIEW 3 of 20 protein 15%-20% of the energy intake. As far as alcohol use is concerned, a moderate intake of ethanol is an additional characteristic of the MD (in particular, wine) [24]. A synthetic example of the recommended composition of a meal in the MD is given in Figure 2.  The MD is therefore rich in macronutrients that have been shown to have a beneficial effect on glucidic and lipidic metabolism and, consequently, on fatty liver disease [13]. In particular, the MD is high in mono-unsaturated fatty acids (MUFAs), poly-unsaturated fatty acids (PUFAs), and fibers,  protein 15%-20% of the energy intake. As far as alcohol use is concerned, a moderate intake of ethanol is an additional characteristic of the MD (in particular, wine) [24]. A synthetic example of the recommended composition of a meal in the MD is given in Figure 2.  The MD is therefore rich in macronutrients that have been shown to have a beneficial effect on glucidic and lipidic metabolism and, consequently, on fatty liver disease [13]. In particular, the MD is high in mono-unsaturated fatty acids (MUFAs), poly-unsaturated fatty acids (PUFAs), and fibers, The MD is therefore rich in macronutrients that have been shown to have a beneficial effect on glucidic and lipidic metabolism and, consequently, on fatty liver disease [13]. In particular, the MD is high in mono-unsaturated fatty acids (MUFAs), poly-unsaturated fatty acids (PUFAs), and fibers, and low in refined sugars and added fructose. MUFAs reduce risk factors for MetS which are linked to NAFLD, such as waist circumference, high-density lipoprotein cholesterol, triglycerides, and glucose, and therefore have a protective effect against cardiovascular events [13]. PUFAs, particularly omega-3 fatty acids, have been shown to be beneficial in preventing cardiovascular events via an improvement of insulin-sensitivity, an anti-inflammatory effect, and a reduction in oxidative stress [25][26][27]. Dietary fibers reduce the risk of MetS components (type 2 diabetes, dyslipidemia) and cardio-vascular risk, and may be involved in a reduction of the risk for NAFLD via a modulation of gut microbiota, thus leading to the increased production of short-chain-fatty acids and phenolic compounds, which have antioxidant properties [28]. These effects are important goals in NAFLD patients since cardiovascular events are the major complication leading to death [29]. Furthermore, it has been shown that hepatic steatosis is reduced in patients consuming high amounts of MUFAs [30]. In particular, in a cross-over study including 12 patients, Ryan et al. found that a MD pattern rich in MUFAs, mainly from olive oil, was associated with a reduction in steatosis (analyzed by magnetic resonance) even if a reduction in body weight was not achieved [31]. This study had the limitation of including a low number of patients but its results were strengthened by the randomized cross-over design and the use of magnetic resonance spectroscopy for the measurement of hepatic steatosis. It needs to be emphasized that the protective effect of this MD pattern may be driven not only by MUFAs, but also by the phenolic compounds in which olive oil is rich, exerting anti-inflammatory, metabolic, and antioxidant effects [32][33][34][35].
PUFAs, and in particular omega-3 fatty acids, exert a similar effect on NAFLD. More precisely, a diet rich in omega-3-fatty acids has been shown to be protective against fatty liver as it prevents hepatic fat accumulation and reduces hepatic steatosis [36][37][38][39][40]. Additionally, it seems to be important that a low omega-6/omega-3 ratio is maintained in order to obtain benefit from omega-3 PUFAs. This evidence comes from observational studies showing higher rates of NAFLD and NASH in patients following a diet with a higher omega-6/omega-3 ratio [41][42][43]. However, no benefits have been described regarding the regression of fibrosis nor the regression of NASH [40,44].
An additional benefit of the MD on fatty liver disease is related to its low content of refined sugars, fructose, and high content of complex carbohydrates and fibers. Epidemiologic reports have identified a directly proportional association between refined sugar, particularly high-fructose corn syrup, and the risk for NAFLD, even in the absence of additional risk factors [45][46][47][48]. Moreover, Western dietary patterns, which are typically rich in added fructose and corn syrup, and added fructose-rich diets have been proven to increase hepatic steatosis in humans and in mice models, whereas a negative correlation was found between low-carbohydrate diets and the risk for hepatic steatosis [49][50][51]. Additionally, it has been widely demonstrated that processed foods, especially soft-drinks, promote liver fat accumulation through de-novo lipogenesis of fructose in the liver, a mechanism that is already altered in NAFLD patients [52][53][54].
Unlike the Western diet, the MD is rich in whole grains, which have a high content of fibers. Whole grains and fibers may be beneficial in NAFLD patients for different reasons. The first is that they have less energy density and induce more satiety than refined carbohydrates. Secondly, they have prebiotic effects that modulate gut microbiota, which may play a role in the pathogenesis of NAFLD and its progression. Finally, they have well known protective effects against the risk for diabetes, dyslipidemia, cardio-vascular diseases, and all-cause mortality [55,56]. Animal models have shown that prebiotic fibers may modulate the human microbiome, leading to a proliferation of bacterial strains that produce short-chain fatty acids with anti-inflammatory properties, and whose metabolism leads to reduced serum cholesterol levels and triglycerides in the liver [28,[57][58][59].
A minimal amount of meat and processed meat intake is another characteristic of the MD. Meat contains cholesterol, saturated fatty acids (SFAs) which are relevant in NAFLD and some case-control and cross-sectional studies have shown the presence of a correlation between meat consumption and NAFLD [41,60].
The MD consents a moderate amount of alcoholic units per day. Although there is a lack of studies with convincing evidence to support the benefits versus harms of ethanol intake, light alcohol intake seems safe in NAFLD non cirrhotic patients [61,62]. Indeed, a recent retrospective study on the effects of alcohol consumption on survival in NAFLD-patients, reported that drinking 0.5-1.5 drinks per day decreases the risk of overall mortality by 41% (hazard ratio (HR) = 0.59, 95% confidence interval (CI) 0.40-0.85, p = 0.005) compared with not drinking. On the contrary, drinking ≥1.5 drinks per day showed a harmful effect [63]. However, this study focused on the effects of alcohol on overall survival and its effects on liver disease progression are not described.
Lastly, there is no specific recommendation about coffee consumption in a typical Mediterranean dietary pattern. However, coffee may have beneficial effects on NAFLD prevention and NASH severity [64][65][66]. Interestingly, Molloy et al. showed that coffee consumption was significantly associated with a reduced prevalence of steatosis and a lower severity NASH. More specifically, when comparing patients with bland steatosis/not-NASH to those with NASH stage 0-1 and also when comparing patients with NASH stage 0-1 to those with NASH stage 2-4, there was a significant difference in coffee consumption between the two groups (p = 0.005 and p = 0.016, respectively). Importantly, this study also reported a significant correlation between coffee consumption and hepatic fibrosis (r = −0.215; p = 0.035) [66]. Notably, an inverse relationship between coffee and HCC risk has been shown [67][68][69]. Indeed a recent metanalysis including twelve studies on HCC (3,414 patients) and six studies on chronic liver disease (1,463 patients) evidenced that the summary relative risks for HCC were 0.66 (95% confidence interval (CI): 0.55-0.78) for regular, 0.78 (95% CI: 0.66-0.91) for low, and 0.50 (95% CI: 0.43-0.58) for high coffee consumption, respectively [70]. Therefore, considering also that evidence supports the cardioprotective effects of coffee and a reduced prevalence of MetS in coffee-consumers [71][72][73], a moderate coffee intake (2-3 cups/day) can safely be recommended.

Current Evidence about the Effects of the Mediterranean Diet on NAFLD
On the basis of the reported benefits of the MD components on the features of the MetS, including NAFLD, the MD has been proposed as the diet of choice for the dietary treatment of hepatic steatosis [74]. Herein, we report the current evidence on the liver-related outcomes of the MD diet. The evidence for the protective effects in terms of cardiovascular events are not the aim of this review and have been extensively reported elsewhere [75]. Indeed, the indication for the MD as a treatment for NAFLD is recent and is based on few observational and interventional studies, of which only two included the performance of a liver biopsy. Most of the studies have focused on assessing the role of MD, and variants of it, on the prevention and reduction of steatosis, as shown in Table 1.

NAFLD Diagnosis and Assessment at Follow Up Baseline Parameters Main Results
Della Corte et al. [84] Observational study Adherence to MD was assessed at time of inclusion To analyze the association between adherence to the MD(using the KIDMED Index) and NAFLD, with laboratory and histologic evaluation, in a group of children and adolescents with obesity. In a case-control study, a Mediterranean dietary and lifestyle pattern was associated to a lower likelihood of having steatosis, independently from body weight and energy intake [85]. The investigators included 100 ultrasound-proven NAFLD patients and 55 healthy controls; dietary habits were assessed through a semi-quantitative food questionnaire. Habitual total night sleep hours as well as naps were reported as well. A significant association was found between a dietary pattern with the characteristics of a MD with a higher consumption of sweets, lower consumption of vegetables and nuts (all p < 0.005), and lower physical activity (p = 0.006) and NAFLD. Likewise, sub-optimal sleep duration was also associated with NAFLD (p = 0.005, 95% CI 0.14-1.01).
In a prospective study, a "Spanish Ketogenic Mediterranean Diet", rich in omega-3 fish oil and olive oil, low in carbohydrates and meat, was prescribed as a treatment in 14 obese men with MetS and NAFLD (diagnosed by ultrasound and ALT levels >40 U/L). Steatosis degree (measured by means of ultrasound scanning using a 4-point validated scale [86]) was significantly reduced in the treatment group (p < 0.001), and complete steatosis remission was achieved in 21.4% of patients with a 92.86% overall reduction; accordingly, a significant reduction of hepatic necrosis enzymes was reported (ALT from 71 to 39 U/L, AST from 47 to 29 U/L; p < 0.001) [76]. Similar results were obtained by Ryan et al. in a pilot cross-over study including 12 obese non-diabetic men with biopsy-proven NAFLD [31]. In this case, all patients undertook a MD and a control diet (low-fat/high carbohydrate diet based on the American Heart Association Diet and Australian National Heart Foundation) in random order for 6 weeks, with a 6 week wash-out period in between. Hepatic fat content was measured by means of magnetic resonance H-spectroscopy, which calculates intrahepatic lipid that is considered the gold standard for quantification of hepatic steatosis. This parameter was significantly improved after the MD treatment period (mean i intrahepatic lipid decreased by approximately 39% from baseline, p < 0.05) as well as insulin-resistance (p < 0.001), even in the absence of weigh reduction. The control diet, surprisingly, failed to achieve such endpoints, and as far as liver enzymes are concerned, no reduction was observed after either dietary intervention; however, this latter finding might be due to the already normal levels of the liver enzymes at enrollment. Another recent randomized controlled trial investigating the effects of the MD diet and a low-fat diet on fatty liver and cardiometabolic risk factors, proved the effectiveness of MD in the reduction of hepatic fat, measured by means of magnetic resonance spectroscopy. Forty-nine subjects with magnetic resonance-proven NAFLD were randomized to a 12-week blinded dietary intervention (MD versus low-fat): at week 12, hepatic steatosis and liver enzymes were significantly reduced in both groups (p < 0.001) and there was no difference in liver fat reduction between the two interventions, with an overall mean reduction of 25.0% [12]. Interestingly, these results were achieved even in the absence of significant weight loss as in this cohort the average weight loss was around 2% from the baseline, which is not the weight loss target (5-7%) recommended by the latest EASL-guidelines.
Additional evidence for the benefit of the MD in fatty liver disease has been reported in a recent randomized clinical trial which was conducted on 50 overweight patients whose liver fat was assessed with ultrasonography and staged by the Hamaguchi score (a highly specific and sensitive scale which uses a 6-point scoring system based on hepato-renal echo contrast, liver brightness, deep attenuation and vascular blurring) [77]. Patients were randomized to three groups (MD alone/MD and antioxidant supplementation with milk thistle/control) and followed a 6-month MD with/without an antioxidant supplement. After 6 months of intervention, a significantly (p = 0.0001) decreased hepatic fat content, assessed by ultrasonography and as compared to baseline, was reported for patients on MD but not for controls. Likely, the fatty liver index (FLI)-which is a validated algorithm (range, 0-100) based on body mass index, waist circumference, triglycerides and gamma-glutamyl-transpeptidase values-decreased significantly [77,87].
The MD pattern is not only characterized by diet but also by regular physical activity so that it can be considered a lifestyle and not a simple dietary pattern. In accordance with this concept, some authors have reported that the beneficial effects of the MD may be mediated more intensely by the MD lifestyle rather than by the diet itself [34,80,88]. Indeed, Abenavoli et al. described a case series including 7 obese patients who were prescribed a strict MD regimen, physical activity and antioxidant supplement (milk thistle): at 6-week evaluation all patients had improved Hamaguchi score, as it ranged from 1-3 at baseline and 0-1 at the end of follow up [89].
Additional evidence for an amelioration of steatosis in patients on a MD lifestyle is provided by the results of a study by Gelli et al. who analyzed the clinical effectiveness of nutritional counseling on reduction of NAFLD severity in 46 adults diagnosed with steatosis by ultrasonography [79]. In this non-controlled study, the percentage of patients with severe steatosis decreased from 52% to 9%, with a remission of steatosis in 20% of the study cohort. More in detail, the fatty liver index decreased from a mean value of 61.55 ± 26.76 to 48.83 ± 31.01 (p < 0.01). Regarding liver enzymes, aspartate and alanine aminotransferase, and gamma-glutamyl-transpeptidase significantly decreased during treatment but the normalization was particularly evident for alanine aminotransferase whose prevalence of alteration decreased from 67% at baseline to 11% following treatment; in particular, aspartate aminotransferase mean values decreased from 36 ± 21 at baseline to 25 ± 11 at 6 months (p < 0.01), while alanine aminotransferase values decreased from 66±31 to 37 ± 19 at 6 months (p < 0.01) and gamma-glutamyl-transpeptidase decreased from 60 ± 61 to 40 ± 35 (p < 0.01). The results of the study also highlighted the benefit of a nutritional counseling which increased the adherence to the dietetic and lifestyle regimen.
Similarly to Gelli et al. also Trovato et al. reported that an intervention focused to increase the adherence to the MD [assessed by the Adherence to Mediterranean Diet Score (AMDS)] and the level of physical exercise significantly decreased hepatic steatosis, measured by Bright Liver Score (BLS) after 6 months in 90 non-diabetic obese patients (BMI 31.0 ± 5.2) [78]. Interestingly, at a multiple linear-regression age-balanced analysis, the changes in AMDS were related to the reduction in hepatic fat content independently of physical activity. However, a potential limitation of this study is the fact that the BLS is not accurate in the staging of fatty liver and is indeed not included among the validated steatosis scores by the most recent EASL/EASD/EASO guidelines [74].
Additional evidence about the effect of a variant of the MD, i.e., a low-glycemic MD, on NAFLD severity, measured by means of ultrasonography was reported by an Italian randomized controlled trial including 98 men and women with moderate or severe steatosis [81]. Low-glycemic MD, not energy-restricted, was effective in the reduction of FLI and alanine aminotransferase levels. More specifically, FLI median and interquantile range significantly decreased from baseline values of 82.28 (70.31-90.38) to 6-month values of 57.72 (27.33-73.14, p < 0.05), while alanine aminotransferase levels decreased from 48 U/L to 39 U/L (p < 0.05) [83].
Interestingly, a recent randomized controlled trial addressed the question whether a reduction in hepatic fat content (HFC) has prognostic significance beyond the loss of visceral adipose tissue [11]. In this study, 278 participants with abdominal obesity or dyslipidemia were randomized to low-fat or Mediterranean/low-carbohydrate (MD/LC + 28 g walnuts/day) diets with or without moderate physical activity. Hepatic fat content and abdominal fat-depots were measured using magnetic resonance imaging. After a 6-and 18-month follow-up period, while the reduction in hepatic fat content was similar between physical activity groups, MED/LC induced a greater proportion of decrease in hepatic fat content (p = 0.036), and a greater improvement in cardiometabolic risk parameters (p < 0.05) than the low-fat diet, even after controlling for visceral adipose tissue changes. Importantly, after controlling for visceral adipose tissue loss, decreased hepatic fat content also remained independently associated with reductions in liver enzymes and glycated hemoglobin. These findings suggest that the beneficial effects of the MD/LC diet on specific cardiometabolic parameters may be mediated more by decreases in hepatic fat content than visceral adipose tissue loss.
Additional evidence about the effects of the MD on fatty liver disease in the mid-and long-term (12 months) will be shown in the near future by the results of the MEDINA study, a randomized controlled trial including 94 patients with NAFLD and insulin-resistance who have been randomized into two groups: MD or low-fat diet [84]. This trial aims to demonstrate, in a large cohort of participants with NALFD, that the MD can result in significant benefits in liver fat and insulin sensitivity and that these changes are sustained at 12 months, independently of weight loss [90].
As the reduction in liver enzymes is concerned, more evidence comes from a recent metanalysis including twenty randomized controlled trials including an overall 1073 NAFLD patients which showed that, compared to standard care-i.e., general advice for healthy diet and lifestyle-a more scheduled exercise and dietetic program improved aminotransferases, hepatic fat, and NAFLD activity score, irrespectively of weight change [81]. Regarding the type of exercise, aerobic compared to resistance exercise did not yield any superior improvements on liver parameters, whereas moderate-to-high volume moderate-intensity continuous training was more beneficial compared to continuous low-to-moderate-volume moderate-intensity training or high intensity interval training [80]. The same group conducted a randomized, controlled, single-blind clinical trial investigating the effect of the MD or of Mediterranean lifestyle in NAFLD patients. Sixty-three overweight or obese patients with ultrasonography-proven NAFLD and elevated alanine aminotransferase and/or gamma-glutamlyl-transpeptidase were randomized to a control group, or to MD or Mediterranean lifestyle (ML) groups. Participants included in the MD and ML arms participated in group sessions aiming at weight loss and increasing adherence to the MD. Additionally the ML was given guidance for increasing physical activity and improving sleep habits, while patients in the control group received only general written information for a healthy lifestyle. At the end of the six-month follow-up period, the ML group showed significant improvements in alanine aminotransferase levels and liver stiffness compared with the control group, while in contrast the MD group failed to achieve these endpoints. Indeed, the ML group achieved significant reduction in alanine aminotransferase levels (p = 0.009) and liver stiffness (p = 0.004) as compared to the control group (after adjusting for percentage of weight loss and baseline values) whereas the MD group improved only liver stiffness compared with the control group (p < 0.001). No differences were noticed regarding the study's primary and secondary outcomes (clinically significant decrease in alanine aminotransferase levels and significant improvements in liver stiffness, NAFLD fibrosis score and gamma-glutamlyl-transpeptidase values) between the MD and ML groups, except that after controlling for baseline values and the proportion of weight loss-patients, the ML group tended to have a higher likelihood of halving their ALT levels compared with the MD group, though not significantly so (OR = 0.27; 95% CI, 0.07-1.03). This evidence suggests that a lifestyle-guided intervention may be the optimal treatment strategy for NAFLD [82].
To the best of our knowledge, the effects of the MD on NASH have been investigated only by two studies [91,92]. Kontogianni et al. conducted an observational study which showed that a higher adherence to the MD was associated with lesser degree of insulin resistance, as measured by the HOMA-score (p = 0.005), and less severe steatosis at histology (p = 0.08) among patients with NAFLD [91]. Moreover, a lower adherence to the MD was associated with a higher risk of progression towards NASH. More in detail, logistic regression analysis revealed that one unit increase in the MedDietScore (a score that assesses the adherence to MD via a questionnaire) was associated with 36% lower likelihood of having NASH, after adjusting for sex and abdominal fat (OR: 0.64; 95% CI, 0.45-0.92, p = 0.02) [83]. Similar results were reported by Della Corte et al. in a cohort of 243 obese pediatric patients [92]. The level of adherence to the MD was evaluated with a questionnaire, the Mediterranean Diet Quality Index for children and adolescents (KIDMED). A low KIDMED was significantly higher in patients with NASH compared to patients without NASH, with fatty liver and without fatty liver (100% versus 29.79% versus 37.88% versus 9.10%, p = 0.01) and correlated with NAFLD activity score >5 and to grade 2 fibrosis [84].
The only study that has described the effects on liver stiffness so far is a prospective study by Abenavoli et al. aiming at the comparison between the effects of the MD with or without an antioxidant complex supplement in overweight patients suffering from NAFLD [89]. The study proved that the MD alone or in association with the antioxidant complex, improved lipid profile, insulin resistance and reduced hepatic fat accumulation and liver stiffness (reductions in transient elastography of approximately 21%-17% in the MD groups were shown whereas the control group showed an increase in liver stiffness by 8.7%, p = 0.0001) [77].
EASL/EASD/EASO NAFLD guidelines recommend diet and physical activity as the best treatment for steatosis. However, a specific training program is not described. Studies investigating the effects of exercise on NAFLD have been conducted with similar results suggesting that both aerobic and resistance training reduce hepatic steatosis [91][92][93][94][95]. Recent metanalysis confirmed this evidence, reporting that independently from which type of exercise program is followed (aerobic vs. resistance) the steatosis grade is significantly reduced if a scheme including at least 3 weekly sessions of 40-45 min for 12weeks is followed [96,97]. Apparently, males tend to have a greater benefit in terms of hepatic fat reduction from aerobic exercise, whereas women have the best benefit from resistance training [96]. Obviously, high compliance to exercise is needed, and a significant proportion of NAFLD patients may have difficulties following aerobic exercise training because of concomitant cardiovascular comorbidities or physical disability due to obesity. Therefore, when considering which training is the best, an individualized decision has to be made, taking into account patients' comorbidities. Lastly, the principal end-point of all the studies assessed reported positive outcomes (i.e., decrease in aminotransferase, improvement in steatosis or histology) in patients who experienced weight loss, and all the studies combined diet and exercise.

Future Research
NAFLD is going to become a major global health concern, and thus there is an increasing need to raise the awareness about the prevention and treatment of this disease, which nowadays seems to be very low [98].
Although weight loss can be an effective treatment, for most patients it is a difficult goal to achieve and maintain. MD has recently been suggested as the diet of choice for NAFLD treatment but the evidence behind this recommendation is very low as most of the studies that have addressed this issue included few patients, differed in the inclusion/exclusion criteria, in the methods by which steatosis was measured, and in the measurement of outcomes.
In order to have approved regimens for the treatment of NASH/NAFLD, therefore, more studies with large cohorts of patients, with consistent clinical endpoints as well as inclusion criteria and methods, are needed. Currently, the resolution of NASH and/or reduction of fibrosis stage are the accepted endpoints by the regulatory authorities since NASH progression and fibrosis stage have prognostic implications. In this context, although histologic NASH has been associated with advanced fibrosis, it is not an independent predictor of long-term mortality. In contrast, there are significant data to suggest that the fibrosis stage is the only robust and independent predictor of liver-related mortality, and it is auspicated that in the future, adequately powered studies will disclose whether the MD may truly represent a viable therapeutic means able to obtain these relevant goals [99,100].