The overriding goal of the treatment of patients is its effectiveness and safety. However, all medications currently being used also exert some adverse pharmaceutical reactions, which may be regarded as an unintended but inevitable cost of pharmacotherapy. The kidney, as the main organ that eliminates xenobiotics, is an organ especially predisposed and vulnerable to the toxic effects of drugs and their metabolites during their excretion from the body. Moreover, some drugs  have a “preferential” nephrotoxicity potential, and their use is associated with an increased risk of kidney damage. Drug nephrotoxicity is, therefore, both a significant problem and a complication of pharmacotherapy. It should be noted that, currently, there is no generally recognized definition of drug-induced nephrotoxicity and no clear criteria for its diagnosis. This review briefly describes the epidemiology and diagnosis of drug-induced nephrotoxicity and characterizes its pathomechanisms, including immunological and inflammatory disturbances, altered kidney blood flow, tubulointerstitial injury, increased lithogenesis–crystal nephropathy, rhabdomyolysis, and thrombotic microangiopathy. The study also lists the basic drugs with nephrotoxicity potential and provides a short overview of the preventive methods for reducing the risk of drug-related kidney damage developing. The kidneys are small organs  located retroperitoneally on the posterior abdominal wall and found between the transverse processes of T12 and L3. Human kidneys account for only about 0.2–0.5% of total body weight, but they receive as much as 20% of the cardiac output. Despite their small size, kidneys are essential for life due to the high renal blood flow value and the role of the kidneys as the main point of elimination of xenobiotics, potentially nephrotoxic substances that are delivered to the organ in relatively high concentrations. Thus, it is reasonable to assume that the kidneys and urinary tract are privileged targets of the noxious actions of drugs and other xenobiotics [Kidneys carry out a lot of integrated functions aimed at maintaining the homeostasis of the body, including: the control of the composition and volume of body fluids; electrolytes and acid-base balance; the long-term regulation of blood pressure in a sodium-dependent manner; and the synthesis and secretion of several hormones  . HoweverDrugs and their metabolites are eliminated from the body in the urine by both glomerular filtration and tubular secretion. The ultrafiltrate formed as a result of glomerular filtration is modified by the tubular transport taking place mostly in the proximal tubules. In the context of nephrotoxicity, it is important that the tubular cell uptake of potentially nephrotoxic compounds occurs via the apical (via endocytosis/pinocytosis and other passive or active transport pathways) or basolateral (via the peritubular capillaries) membranes of the proximal tubules. In the process of tubular uptake, the family of active transporters, being organic anion transporters (OAT) for negatively charged drugs and organic cation transporters (OCT) for positively charged drugs, as well as sodium dicarboxylate transporters or other active transport pumps, are involved . OverallDrug-induced nephrotoxicity, also less frequently named drug-induced kidney disease (DIKD), is a common clinical problem. It is estimated to affect as many as 14–26% of adult and 16% of pediatric patients . HoweverAlthough there is no standard definition, DIKD is defined as any damage to the kidneys caused directly or indirectly by drugs, which leads to kidney dysfunction. DIKD is usually classified according to the predominant pathomechanism responsible for kidney damage development or based on the time of the course, with the latter division including both acute and chronic DIKD forms. Acute DIKD is recognized as a 0.5 mg/dL or 50% rise in serum creatinine over a 24–72 h time frame after a minimum of 24–48 h of drug exposure ,11. BaseConsidering the problem of the difficult clinical description of drug nephrotoxicity and a lack of an unequivocal consensus on its diagnosis, a novel framework of drug-related nephrotoxicity diagnostic and therapeutic procedures has been proposed by Awdishu and Mehta, which focuses on risk assessment, early recognition, targeted response, timely renal support and rehabilitation, and research into the epidemiology and pathophysiology—otherwise known as the rules of the “6R’s of drug-induced nephrotoxicity” . They arRisk: Risk factors for drug-induced nephrotoxicity include patient, kidney, and drug-related factors. They are listed in Recognition: In order to improve the diagnosis of kidney drug-related dysfunction, it was assumed that the phenomenon may take the form of one of four phenotypes based on clinical presentation, these being acute kidney injury, glomerular disturbances, tubular disturbances, and nephrolithiasis. Primary and secondary clinical criteria should be revealed to support the specific phenotype recognition, and in the case of acute kidney injury, acute kidney disease, and chronic kidney disease, an appropriate timeframe based on the KDIGO findings characteristic for these diseases should also be demonstrated ,12. The In each of the particular phenotypes listed in Response: The response to the treatment depends on the phenotype, the severity of the injury, and the underlying disease indicative of the particular drug. Therefore, during the treatment of nephrotoxicity, concurrent risk factors for kidney injury must be taken into account, such as hypertension, hyperglycemia, and anemia; in order to minimalize the possible drug interactions that could worsen kidney damage .Renal support: Implementing kidney support  during the treatment of kidney dysfunction enables the removal of the offending drug from the blood, minimalizing the resulting damage and supporting the kidneys in achieving their recovery. However, the decision to subject a patient to dialysis/hemodialysis in the course of drug-induced kidney dysfunction is complex, and the procedure is usually only implemented in patients with severe kidney damage and in the case of nephrotoxic drugs, characterized by a sufficiently high free-fraction that can be removed by dialysis .Renal rehabilitation: Rehabilitation and the clinical monitoring of patients presenting drug-induced nephrotoxicity symptoms involve the repeated evaluation of the kidney functions in order to assess the potential reversibility and to avoid re-exposure to the offending drug. Furthermore, the aim is to report all of the adverse effects that may have occurred during the administration of potentially nephrotoxic agents. A valuable diagnostic tool facilitating the individualization of treatment and contributing to improving the safety of therapy is therapeutic drug monitoring (TDM) in the blood. The procedure should be employed, if available, as it may prevent the further development of nephrotoxicity . As alreResearch: The last “R” in the approach proposed by Awdishu and Mehta  is reseaIn addition to the above-mentioned classification, based on the 4 main drug-induced nephrotoxicity phenotypes proposed by Awdishu and Mehta , there iIn general, several mechanisms underlying the development of drug-related nephrotoxicity can be distinguished: inflammatory and immune-related mechanisms leading to acute or chronic glomerulonephritis or interstitial nephritis; altered intraglomerular hemodynamics, triggering acute kidney injury; tubular cell toxicity with the risk of acute tubular necrosis; crystal nephropathy; and rhabdomyolysis or thrombotic microangiopathy.Inflammatory and immune mechanisms: Drugs can cause inflammatory and immune reactions in all segments of the nephrons, such as in the glomerulus, tubular cells, and the interstitium. The immunocomplex and hapten-mediated mechanisms may be recognized, and drugs can induce the production of antibodies against them and form immune complexes. These complexes can deposit in the glomeruli, contributing to glomerular damage (glomerulonephritis), but also “leak” into the interstitial microcirculation, binding to the basal membrane of the renal tubules, initiating interstitial inflammation. The most reported causative agents for glomerulonephritis are gold compounds, hydralazine, lithium, propylthiouracil, and non-steroidal anti-inflammatory drugs (NSAIDs) . Some drThe so-called Chinese herb nephropathy was reported for the first time in young Belgian women who were using Chinese herbal slimming remedies. Since then, patients with aristolochic acid nephropathy have been reported worldwide. In vivo and in vitro experiments have revealed some mechanisms of aristolochic acid nephropathy, such as endoplasmic reticulum stress and injury, increasing oxidative stress injury, initiation and sustaining an immune-mediated inflammatory mechanism, and kidney tubular epithelial cell transdifferentiation. Microscopically, the typical findings involve extensive interstitial fibrosis with atrophy and loss of tubules located in the superficial cortex and progressing toward the inner cortex. The interstitium is usually significantly hypocellular, and glomerular lesions mainly include ischemic, microcystic, obsolescent glomeruli, occasional thrombotic microangiopathy-like lesions, and/or focal segmental sclerosis-like lesions. Moreover, apart from nephrotoxicity, aristocholic acid has been proven to be implicated in the genesis of urothelial carcinoma occurring in the renal pelvis and upper ureter ,29.The chronic, long-term administration of non-opioid analgesics  is linked with an increased risk of chronic tubulointerstitial nephritis development. The chronic ingestion of these drugs in high doses (more than 1 g daily for more than two years) is believed to result in chronic interstitial inflammatory changes that disrupt the vascular system causing ischemia and leading to scarring and fibrosis. The outcome is papillary necrosis and CKD development ,19,20,21Altered intraglomerular hemodynamics and acute kidney injury: Glomerular filtration is a result of glomerular blood flow. The size of the kidney filtration fraction is determined by the effective arterial blood volume and the regulation of the vascular resistance of the afferent and efferent arterioles. The relaxation of the glomerular afferent arterioles and the contraction of the efferent arterioles provide for the maintaining of, and the autoregulation of, intraglomerular pressure and preserving GFR and urine output. The relaxation of afferent arterioles is controlled by the prostaglandins, while the contraction of the efferent ones is regulated by angiotensin II ,37. ThusTaking into account drugs interfering with the RAA system, it should be emphasized that both ACEI and ARB are seldom truly nephrotoxic, although their effect on intrarenal hemodynamics can cause a reversible reduction in the glomerular filtration rate (GFR), without structural damage, and eventually, ultimately AKI development. Such a damaging effect on the kidneys is potentially possible in patients with an already existing impairment of glomerular autoregulation, based on the prostaglandin-mediated vasodilation of the afferent arteriole and angiotensin-mediated vasoconstriction of the efferent arteriole; congestive cardiac failure, chronic kidney disease, or in the setting of reduced renal perfusion  . On the The nephrotoxicity potential of ACEIs/ARBs may also result from drug interactions. The simultaneous chronic use of ACEI/ARB and NSAID is an example of possible drug interaction with a high potential for nephrotoxicity, especially in elderly patients. The risk is even increased when a diuretic agent is also administered (“triple whammy interaction”). One observational study analyzed the frequency of adverse reactions in patients over 75 years of age with multiple diseases receiving polypharmacotherapy. In the study, 12 patients were prescribed the ACEI/NSAID combination. It was then revealed that two of them developed acute renal failure, of whom one died and the other recovered after the discontinuation of both drugs. Moreover, four patients showed deterioration in renal function, which returned to normal after at least one of the drugs was stopped. Renal function remained stable in six patients. Therefore, it can be concluded that in elderly patients, ACEI and NSAID should not be used chronically, especially with an additional combination of diuretics . In anotMoreover, some drugs  may lead to the vasoconstriction of the afferent vessels in a prostaglandin-independent manner, also leading to kidney function impairment ,19,20,21Tubular cell toxicity and acute tubular necrosis: As mentioned in the introduction, the proximal tubules are particularly vulnerable to the effects of nephrotoxins, including drugs. Drug-related tubular injury is triggered after drug reabsorption and their entering the tubular cells. Intracellularly, drugs may cause mitochondrial dysfunction, which impairs the energy management in the tubular cells, disrupting their integrity and adherence to the basement membrane. There is also the over-expression of adhesive proteins and increased oxidative stress. The increase in intracellular calcium concentration activates the proteases, which initiates the pathway involved in inflammation development and cell death through apoptosis or necrosis. Moreover, the transport functions of cells are disturbed, and the filtrate leaks back from the tubular lumen into the interstitial tissue. Ultimately, the tubular cells are exfoliated into the lumen of the tubules and form obstructive conglomerates, which increase intra-tubular pressure and inhibit GFR. Finally, a complex set of pathophysiological disorders known as acute tubular necrosis (ATN) develops. Drugs that are considered to exert their complex nephrotoxic action in such a way include aminoglycosides, amphotericin B, antiretroviral drugs, cis-platinum, iodinated contrast agents, foscarnet, and bisphosphonates ,19,20,21It is estimated that up to 25% of all patients treated with aminoglycoside develop nephrotoxicity. Aminoglycosides exert their noxious effects as renal tubular toxicity, reduced glomerular filtration, and a reduction in renal blood flow. Aminoglycosides are polycationic agents that are subject to apical tubular uptake from the urinary space via endocytosis/pinocytosis after binding to megalin/cubulin receptors. These drugs are then translocated into lysosomes (followed by the inhibition of degrading enzymes), the Golgi body, the endoplasmic reticulum, and inside the mitochondria where they induce apoptosis and necrosis. Lysosomal injury is associated with membrane and organelles damage (“myeloid bodies”), increased oxidative stress, and mitochondrial dysfunction. Finally, energy failure in the tubules develops and contributes to the loss of their integrity and disturbances of numerous transmembrane transporters impairing tubular reabsorption. In the glomerulus, aminoglycosides increase intracellular calcium levels and induce mesangial smooth muscle contraction, which leads to decreased GFR. The reduction in renal blood flow is caused by increased vascular resistance in the renal vascular bed, resulting from endothelin and thromboxane release ,44,45.Amphotericin B is an anti-fungal agent used in many systemic infections, and this drug has high nephrotoxic potential. The mechanism of kidney damage induced by amphotericin B involves directly affecting the cell membrane resulting in increased permeability, as well as indirect effects produced by the activation of intrarenal mechanisms (tubuloglomerular feedback), the release of mediators (thromboxane A2), and changes in intracellular calcium levels. The most known consequences of amphotericin B therapy are tubular damage and the acute decrease of renal blood flow and filtration rate leading to AKI ,47.Antiretroviral drugs are another example of tubulopathy-potential agents. Treatment with acyclovir, foscarnet, tenofovir, adefovir, cidofovir and didanosine, lamivudine, stavudine, or zidovudine have all been associated with a risk of ATN development in HIV-infected patients. On the other hand, the administration of protease inhibitors  is associated with the risk of intratubular precipitation of drug metabolites, due to their poor solubility ,49.Tenofovir, as a nucleotide reverse transcriptase inhibitor, is a substrate for the organic anion transporter 1 (OAT-1) located in the proximal tubular basolateral membrane. This drug accumulates in the proximal tubular cells and causes mitochondrial toxicity, likely through the depletion of mitochondrial DNA, leading to energetic failure, cellular dysfunction, and/or death. Consequently, proximal tubule damage is the most recognized form of the nephrotoxicity of antiretroviral drugs, and this dysfunction usually improves after tenofovir withdrawal. However, only 40% of patients achieve complete renal recovery .Cisplatin enters the renal tubules after uptake by basolateral transporters , and intracellularly, it is able to activate signaling pathways leading to cell apoptosis . Moreover, cisplatin causes increased oxidative stress and induces TNF-α production in the tubular cells, which triggers a robust activation of intrinsic and extrinsic apoptotic cascades and endonucleases. Many of these same pathways contribute to the cytotoxic actions of cisplatin in tumor cells. Cisplatin may also induce vasocontraction in the renal vasculature, leading to ischemic tubular cell death and a decreased glomerular filtration rate (GFR). Taking all of the above issues together, these pathological events may result in AKI development ,52.Iodine contrast agents are significant nephrotoxic compounds. Estimates indicate that they account for up to 11% of AKI episodes in hospitalized patients, especially those with pre-existing renal dysfunction or diabetes mellitus, in whom a standard hydration protocol was not administered during imaging ,54. DespBisphosphonates are regarded to be the class of drugs with an attributed low incidence of adverse kidney effects. After dosage, between 27% and 62% of these drugs bind to bone minerals, and the remainder is not metabolized, being excreted via the kidneys; predominantly by both passive glomerular filtration and active transport in renal proximal tubular cells. Although bisphosphonates are generally well tolerated, the demonstration of their kidney adverse events, revealed in postmarketing clinical studies, has resulted in the inclusion of “warnings” on the prescribing information of all bisphosphonates, regarding the use of these agents in patients with severe renal impairment (CrCl <35–30 mL/min). The nephrotoxicity of bisphosphonates (predominantly induced by zoledronate or pamidronate) is mainly dependent on the route of administration; given orally, bisphosphonates are not associated with significant nephrotoxicity, however, nephrotoxicity is a potential limiting factor to the use of intravenous (IV) bisphosphonates, and their nephrotoxicity is both dose-dependent and infusion time-dependent. Pamidronate use has been associated with nephropathy based on focal segmental glomerulosclerosis, whereas zoledronate has mostly been associated with direct tubular toxic effects ,57,58.Crystal nephropathy: Kidney damage may also result from the precipitation and deposition of insoluble compounds, forming crystals and fine concretions within the tubular fluid, after exceeding their solubility; this, however, depends on the urinary concentration of the poorly soluble drug and its metabolites, as well as the urinary pH. This phenomenon occurs mainly in the distal tubules and initiates secondary changes in the renal interstitium. Drugs regarded as being associated with an increased risk of the ability to form intrarenal crystal deposition mostly include sulphonamides, triamterene, antiretroviral drugs used in HAART therapy , antacids , methotrexate, quinolones , and some antibiotics  ,19,20,21Rhabdomyolysis: Rhabdomyolysis is defined as a pathological condition of skeletal muscle cell damage, leading to the release of toxic intracellular material into blood circulation. The key pathophysiological finding in this disorder is an increase of intracellular free ionized calcium due to either cellular energy depletion or direct plasma membrane rupture. Initially, the increased calcium level contributes to the intensification of the skeletal muscle cell contractility. Finally, the consequence of the increased intracellular calcium is the activation of several proteases, mitochondrial dysfunction, and increases in the production of reactive oxygen species, ultimately resulting in skeletal muscle cell death and release of their content into the blood . ClinicaTo sum up, any drug which directly impairs the production or use of adenosine triphosphate (ATP) in the skeletal muscle, or increases their energy requirements, which cannot be compensated for due to inefficient production, or alters calcium metabolism by the sarcoplasmic reticulum, may be an etiological factor of rhabdomyolysis. In addition, drug-induced indirect mechanisms of rhabdomyolysis may be distinguished, including ischemia, as a result of prolonged immobilization from drug overdosage with CNS depressants, thus impairing the delivery of oxygen and nutrients, drug-induced delirium, choreoathetosis, or dystonic reactions and seizures (which increase muscle activity and the demand for ATP) . Thus, dThrombotic microangiopathy: Thrombotic microangiopathy (TMA) is a clinical entity resulting in excessive and uncontrolled thrombosis in capillaries and arterioles induced by endothelial injury. TMA encompasses a heterogenous group of disorders characterized by microangiopathic hemolytic anemia, thrombocytopenia, and microthrombi leading to occlusive vascular thrombosis and ischemic damage or even infarction of the end organ . The primary forms of TMA are thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS) ,70. DrugFor many drugs, the molecular mechanism of their nephrotoxic action has been clarified. The common, complex, interconnected elements of nephrotoxic action at the molecular level involve the activation of signal transducers and intracellular messengers, DNA damage, mitochondrial dysfunction, oxidative stress, sustained inflammatory response, and the activation of apoptotic pathways. Pathomechanisms of nephrotoxicity at the molecular level can be traced to the example of cisplatin. It seems that cisplatin-induced kidney damage has been described in the largest and most detailed way.Cisplatin is predominantly excreted by the kidneys and the drug accumulates in the kidneys. This drug affects mostly proximal tubules, and glomeruli and distal tubules are affected subsequently. The concentration of the drug in tubular epithelial cells is five times greater than in blood. Therefore, even non-toxic blood levels may reach toxic levels in the kidneys. After filtrating into the urine, cisplatin enters the tubular cells via passive diffusion or transport-mediated facilitated diffusion with the participation of basolateral organic cation transporters (OCT), and reaches high concentrations in the proximal tubular cells of the inner renal cortex and outer medulla (S3 segment). Inside the tubular cells, cisplatin forms cross-links within and between DNA chains leading to the activation of various cellular responses, including the signaling of DNA damage, cell cycle checkpoints, DNA repair, and cell death. The drug also accumulates in the mitochondria of tubular cells and impairs mitochondrial bioenergetics due to the inducement of oxidative stress, and the release of pro-apoptotic factors, which ultimately lead to renal tubular cell death ,74,75. ASimilar molecular abnormalities to those seen with cisplatin nephrotoxicity have been reported for other drugs, e.g., aminoglycosides , amphotericin B, antiviral agents  or radiocontrasts because those agents are responsible for AKI development, in the form of acute tubular necrosis, with oxidative stress and necroinflammation as the main underlying mechanisms .In conclusion, taking into account the molecular description of drug-induced kidney damage, it is important to emphasize the importance of the inflammatory process and the increased oxidative stress ongoing in the kidney tissues, with the release of mediators  sustaining the course of inflammatory reactions and as a consequence of pathological damage and tissue remodeling.The most important and obvious preventive factor in reducing the incidence of drug-induced kidney dysfunction is the knowledge of the nephrotoxic potential of the drugs used in the treatment of the patient and the ability to predict the consequences of pharmacotherapy on kidney function. The early and proper recognition of nephrotoxicity symptoms is of key importance and allows for rapid therapeutic intervention and even the withdrawal of the drug used so far. Drug-induced nephrotoxicity, similarly to other kidney diseases, can be diagnosed with simple blood and/or urinary tests, including blood creatinine and urea nitrogen measurement; the estimation of GFR by creatinine clearance; and qualitative and quantitative evaluation of the blood/urine for new biomarkers indicating kidney function. So far, the greatest experience in experimental research, in a clinical setting, is associated with selected protein markers such as cystatin C (a marker of GFR), nephrin, podocin , kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin-1 (NGAL-1), liver type fatty acid binding protein (FABP), and osteopontin . These laboratory parameters are increasingly used in the diagnosis of either AKI or CKD. Moreover, certain markers such as asymmetric dimethylarginine, inflammatory/fibrosis parameters , and Klotho-FGF23 axis raise the most interest as the best selective markers of CKD. A detailed description of the diagnostic application of these markers is beyond the scope of this paper and can be found in numerous reviews dealing specifically with this issue ,89,90,91In clinical assessment, drug-related nephrotoxicity can manifest with AKI , nephrotic syndrome, renal tubular dysfunction, or CKD symptoms. Thus, it is necessary to carefully assess the clinical symptoms appearing in the patient because many kidney disturbances in the initial phases are mildly symptomatic and may be overlooked. General principles for reducing drug-induced nephrotoxicity involve identifying patients at increased cumulative risk of nephrotoxicity and then continuously monitoring kidney function in treated patients, especially when introducing any drug to a patient that is known to be nephrotoxic ,96.The most important issue of safe pharmacotherapy is the individualization of drug dosage, reducing the risk of adverse drug reactions, including kidney damage. Hence, it is crucial to precisely determine the doses of drugs used in the patient, adjusting them to kidney and liver functions. This is possible based on the determination of the individual GFR values in each patient undergoing pharmacotherapy. It is also worth mentioning that most drugs eliminated in the urine do not require dosage adjustment until the creatinine clearance declines below 50 mL/min . The basIf it is necessary to use drugs with significant potential nephrotoxicity, detailed recommendations are developed to prevent their harmful effects on the kidneys. For example, reference can be made to the prophylactic use of antioxidant compounds  that reduce free radical damage in the course of the use of drugs that increase oxidative stress in kidney tissues, e.g., during treatment with vancomycin , cyclophPharmacotherapy implemented for the patient must be effective, but also safe. Kidney damage may be an unintended but possible side-effect of treatment due to this organ being preferentially vulnerable to damage due to exposure to drugs and metabolites during their excretion stage. Hence, the development of drug-induced kidney damage is a possible complication, especially in patients with pre-existing kidney dysfunction and other risk factors. The risk of drug-related nephrotoxicity significantly increases in patients with either primary kidney diseases or numerous diseases outside the kidneys and undergoing polypharmacy with numerous interacting drugs. Each treatment must be individualized, with precise dosage selection based on the functional status of the patient’s kidneys and liver, and covered by the continuous monitoring of renal function, especially in the case of administering drugs with proven nephrotoxic effects, as discussed in this paper. It should be emphasized that some drugs may induce a nephrotoxic effect using several complementary mechanisms . Therefore, the use of many drugs, especially when administered chronically and in high doses, significantly increases the risk of kidney damage. Some substances, such as contrast agents (iodinated and gadolinium), lithium salts, cis-platinum, cyclosporin A, aminoglycosides, and amphotericin B, are characterized by a high intrinsic nephrotoxicity potential, and in the presence of such substances, particular vigilance and continuous monitoring of kidney function are required.


Obstructive sleep apnea (OSA) may result in severe health onditions, reduces quality of live, and affects high percentages of the adult population. Due to recent changes in the German health care regulations, mandibular advancement devices (MAD) will become available as a treatment option for OSA to a greater extent for general dentists and their patients.A guideline development group consisting of nine members representing four German dental and medical organizations was formed, in order to provide critical information and orientation to the main stakeholders (dentists and patients), regarding the use of MAD for the treatment of OSA within dental sleep medicine.This guideline aims to inform physicians and dentists, particularly those with acquired qualification/specialization in sleep medicine (or in the diagnosis and treatment of sleep-related breathing disorders), as well as experts, payers, and patients. It delivers recommendations on technical requirements for MAD prescription and fabrication, clinical procedures, maintenance, and follow-up procedures.A MAD should be designed for long-term therapy and must be a custom made, adjustable, bimaxillary retained two-splint system equipped with adjustable protrusive elements. The fabrication in a dental laboratory should be based on dental impressions or scans and three-dimensional registrations of the starting position taken with a bite gauge. While initial studies reported the prevalence of obstructive sleep apnea (OSA) to be 4% of the male population and 2% of the female population in the USA , recent Treatment recommendations vary, depending on the severity of OSA and snoring, from general measures such as weight reduction, exercise or positional therapy, to more specific treatments such as positive airway pressure (PAP) therapy, occlusal splint therapy with a mandibular advancement device (MAD), and surgical procedures . CompareMADs are used in adults for the management of snoring, OSA, and of the health and social impairments associated with these sleep disorders. Depending on the specific constellation of medical findings, mandibular advancement devices may be used alone or in combination with other treatment options such as diet for obesity, positional therapy for positional OSA and snoring, in combination with positive airway pressure therapy in order to lower the therapeutic PAP-pressure, as well as with surgical methods.Any oral appliance used for MAD for treatment of OSA should be a custom-made, adjustable, bimaxillary retained, two-splint (bibloc) system with adjustable protrusive elements fabricated in a dental laboratory based on dental impressions or scans and three-dimensional registrations of the starting position taken with a bite gauge. The mechanism of action of MAD therapy is predominantly induced by mandibular protrusion, which places tension on the suprahyoid tissues, resulting in luminal enlargement and stabilization of the airway at the level of the velum, the tongue base, and the epiglottis . Due to AWMF-Regelwerk Leitlinien, Version 1.1 of 27 March 2013).The guideline development group consisted of four experts representing the following organizations: German Dental Association, German Society of Craniomandibular Function and Disorders, Orofacial Pain Working Group of the German Pain Society, the German Association of Statutory Health Insurance Dentists, and five experts representing the German Society of Dental Sleep Medicine. The methodology used for the development of this guideline is based on the AWMF Guideline on Guideline Development   , 11–14.Insufficient stability of remaining teeth/insufficient occlusal support zones ;Temporary removable of partial dentures  or permanent removable, fixed, and combined dentures ;Dental caries, insufficient fillings, or other defects ;Periodontitis/periimplantitis  ;Functional disorders of the masticatory system: increased risk in case of function-limiting and/or painful temporomandibular disorders (TMD), especially dysfunctional pain  and function-limiting mandibular hypomobility in the presence of < 5 mm of mandibular protrusion, measured from the maximum retruded position as the best of three attempts, in accordance with the Diagnostic Criteria for Temporomandibular Disorders (DC/TMD) .Clinical findings that may preclude a dental indication for MAD include:The patient’s individual profile of the aforementioned risk factors relevant to treatment decision-making may limit the indications and possibilities for MAD therapy, in the dentist’s assessment.According to the above clinical findings, dentists determine whether and which dental measures need to be initiated before and during OAT for risk reduction, including a possible adjustment of a preexisting MAD.Existing risk factor assessment tools which have been modified in order to capture the treatment-relevant findings and constellations of findings associated with OAT, e.g., vector diagram-type instruments used in dentistry, may be employed , 17. If To ensure optimal fit and function of the oral appliance, precise impressions of the upper and lower arches form the basis for MAD fabrication. These may be conventional or digital impressions, according to the practitioner’s preferences. When taking the impressions, particular attention must be paid to adequate undercut and soft tissue rendering. Clinical parameters that can impair impression quality  must also be taken into account.The vertical dimension of the MAD starting position  remains controversial. There is no clear scientific evidence regarding the optimal vertical dimension and its effect on sleep-related breathing disorders. However, clinical experience has shown that the measured absolute protrusion of the mandible and adherence to MAD therapy decrease with increasing vertical dimension –21.To prevent fracture of the device during treatment, the vertical distance in the posterior region should be large enough to enable adequate dental laboratory work on the respective MAD. The impact of a sharply developed curve of Spee on the interocclusal distance needs to be considered over the complete protrusive course of the mandible.To prevent interference over the total/complete distance of mandibular protrusion, the vertical distance in the anterior region should be large enough to enable adequate dental laboratory work on the specific MAD, but not too large as to impede lip closure during sleep.comfortable (pain- and tension-free) andapproximately 50% of maximum protrusion, if feasible .Recommendations regarding the sagittal dimension of the starting position of MAD therapy (retrusion/protrusion) vary greatly (from 6 to 90% of the maximum protrusion capacity) due to discrepancies in the reference point of measurement, among other things –25. In cThe horizontal dimension of the starting position of MAD therapy (transverse axis) should take the patient-specific lateral deviation of the mandible that occurs during advancement into account and should be comfortable for the patient Table .Table 4RIn order to obtain a reproducible maxillomandibular relation record for the starting position of OAT, dentists should use appropriate registration instruments (bite gauge). The guideline working group recommends the use of registration instruments that permit reproducible adjustment in the three spatial dimensions in a lying and relaxed treatment position, making it easier to obtain reproducible maximum active protrusion and retrusion determinations and painless and tension-free maxillomandibular relationship records.Different technical features of MAD serve to advance the mandible to a more forward position and, thus, expand the upper airway. There are two basic MAD design types: non-adjustable one-piece (monobloc) appliances and adjustable two-piece (bibloc) appliances. Based on the current scientific evidence, adjustable bibloc MADs with specific features should be used in adults , 28–32 .Recent research certifies positive treatment outcomes for titratable and non-titratable MADs regarding sleep parameters in patients with mild to moderate OSA . Due to Appliance delivery involves a number of steps required not only in order to ensure the effectiveness of OAT, the longevity of the MAD appliance, and patient adherence, but also in order to minimize the potential side effects of OAT. Integrity and technical design quality of the MAD have to be controlled by the dentist before initial insertion. Next, the appliance is inserted in the patient’s mouth and checked for fit, retention, and lip closure. , 35.The patient must be informed and instructed about the protocol for MAD titration and adjustment. Patients with a diagnosis of OSA must be informed in advance about the need for a sleep medicine physician to assess the treatment effect of the MAD on completion of the titration phase.Patients must be informed about potential side effects before the start of OAT and also during the course of treatment, if necessary. Jaw exercises must be demonstrated to and practiced with the patient in order to prevent or reduce pain in the jaw muscle and temporomandibular joint areas as well as to ensure the complete reposition of the mandible to its habitual position after removal of the MAD and, thus, to increase patient adherence –38 Tabl.Table 5Rtitration of the MAD, in accordance with dental sleep medicine requirements. The titration phase starts after a successful acclimatization phase; it consists of adjusting the degree of mandibular advancement  and, if necessary in order to reduce side effects, also a horizontal and or vertical adjustment. The aim of these adjustments is to optimize the MAD effect while minimizing the side effects of OAT, which leads to better adherence to the treatment. As the degree of mandibular advancement increases, increased effectiveness of treatment becomes more likely, but there is no linear relationship between the degree of mandibular advancement/readjustment and the effectiveness of MAD therapy [Adjustment of a mandibular advancement device during the initial OAT phase is defined as  therapy , 39. Dat therapy . MetroloThe titration phase ends when the therapeutic position of the MAD has been reached. At this phase, no further adjustment of mandibular position is needed, the patient is satisfied with the result, and the sleep physician has demonstrated that the MAD has a sufficient effect during sleep (sufficient response to MAD therapy) usually after polysomnographic recordings. The MAD should be worn during the entire duration of sleep. As standard procedure, the first dental recall appointment (check-up) should be scheduled 6 months after the end of the titration phase, then annually. Recall intervals can be adjusted in accordance with the individual risk profile of the patient  (Table 6Assessment of the outcome of MAD therapy includes an evaluation of patient-related aspects such as efficacy, manageability, comfort, side effects, and patient adherence to the treatment. Technical aspects of the assessment of OAT quality are related to the MAD: these are fit, adjustability and titratability, durability, convertibility/repairability, cleanability/hygienics and material biocompatibility Table .Table 7The risks and benefits of MAD therapy need to be evaluated from medical and dental perspectives in using an interdisciplinary approach. MAD providers should have sound knowledge about specific appliances and their therapeutic application. MADs should be designed for long-term therapy and must be a custom made, adjustable, bimaxillary retained two-splint system with adjustable protrusive elements, fabricated in a dental laboratory according to dental impressions or scans and three-dimensional registrations of the starting position taken with bite gauge. Explanation of potential side effects of MAD should be given before the start of treatment and instructions on MAD handling during the delivery appointment. Finally, the effectiveness of the therapeutic position of the MAD should be evaluated and confirmed by the sleep physician.


Anthocyanins are extensively used as natural non-toxic compounds in the food industry due to their unique biological properties. However, the instability of anthocyanins greatly affects their industrial application. Studies related to acylated anthocyanins with higher stability and increased solubility in organic solvents have shown that the acylation of anthocyanins can improve the stability and fat solubility of anthocyanins. However, relevant developments in research regarding the mechanisms of acylation and applications of acylated anthocyanins are scarcely reviewed. This review aims to provide an overview of the mechanisms of acylation and the applications of acylated anthocyanins in the food industry. In the review, acylation methods, including biosynthesis, semi-biosynthesis, and chemical and enzymatic acylation, are elaborated, physicochemical properties and biological activities of acylated anthocyanins are highlighted, and their application as colourants, functionalizing agents, intelligent indicators, and novel packaging materials in the food industry are summarized. The limitations encountered in the preparation of acylated anthocyanins and future prospects, their applications are also presented. Acylated anthocyanins present potential alternatives to anthocyanins in the food industry due to their functions and advantages as compared with non-acylated analogues. It is hoped that this review will offer further information on the effective synthesis and encourage commercialization of acylated anthocyanins in the food industry. Anthocyanins are glycosylated derivatives of flavylium cations (2-phenylbenzopyrilium) with different methoxyl or hydroxyl substitutes on the rings, separated by an oxygen-containing six-membered heterocyclic ring ,2. They Acylated anthocyanins present potential alternatives to anthocyanins due to their unique functions and advantages compared with non-acylated analogues, attributed to the structural change in anthocyanins that is a result from the connection of acyl groups ,10,11. AIn order to better utilize acylated anthocyanins, there is a need for a comprehensive understanding of their formation from the perspectives of biosynthesis and chemical synthesis. Fortunately, with the continuous development of genetic engineering, molecular technology, and analytical chemistry, it is possible to better understand acylated anthocyanins at the level of gene expression and chemical synthesis. Proper understanding of the underlying mechanisms of acylation can facilitate the production and application of acylated anthocyanins in vivo and in vitro.There are a few studies reported on the acylation and applications of anthocyanins. Giusti and Wrolstad  reviewedAcylation is the esterification of hydroxyl groups by aliphatic acyl donors or aromatic acyl donors and constitutes of a universally observed modification of plant secondary metabolites, contributing to various products with changeable physical and biological properties . AcylateDue to the unique role of acyltransferase in the acylation of anthocyanins, research is geared towards identifying the key genes encoding acyltransferases for a proper understanding of the entire acylation process, and several key genes that encode acyltransferases in some plants have been identified with the help of genetic engineering and molecular biology. For the acylation via the biosynthetic approach in Arabidopsis, Luo et al.  incorporSeveral other studies have also been reported regarding key genes to encode acyltransferase in other plant tissues. In purple carrot, galactosyl was transferred from UDP-galactose to cyanidin by cyanidin galactosyltransferase , which enabled the anthocyanins to undergo glycosylation and acylation to form stable acylated anthocyanins. The acyltransferase involved in the acylation was produced by the expression of the DcSAT1 gene ,31. MoreVitis davidii) to compare the difference in their transcriptional levels and look for several key genes associated with anthocyanin accumulation in Vitis davidii. They inferred from the results of the transcriptomic analysis that the gene 3AT (VIT-03s0017g00870) played a crucial role in the anthocyanin acylation process. Furthermore, based on the fact that anthocyanin acyltransferases belong to the BAHD family of proteins, Rinaldo et al. [In berry, Sun et al.  conducteo et al.  establisIn a recent study, tobacco lines were successfully developed by modifying key genes in acylated anthocyanin biosynthesis , therebySo far, a complete mechanism of acylation suitable for all kinds of plants is not available. However, the mechanism of acylation follows a general rule of synthesizing three common anthocyanins, followed by the modification of the anthocyanins via glycosylation and then acylation to obtain acylated anthocyanins under the control of glycosyltransferases and acyltransferases. Therefore, knowledge about the key regulatory genes for glycosyltransferases and acyltransferases can provide theoretical guidance for the commercial production of acylated anthocyanins.The process of obtaining acylated anthocyanins via biosynthesis is a cumbersome method that involves a series of operations to identify and express the key regulatory genes. To solve this problem, the semi-biosynthetic method, which combines biosynthesis and chemical synthesis, has been proposed. The mechanism of the acylation of anthocyanins through semi-biosynthesis is mainly employed to synthesize non-acylated anthocyanins in plant cells through biosynthesis and then use acyl donors from the external environment to further synthesize specific acylated anthocyanins. The plant cell suspension culture process is the most commonly used semi-biosynthesis method for obtaining acylated anthocyanins . AccordiWhittemore et al.  obtained−1m−2 and with different concentrations of phenylalanine and methyl jasmonate. Their results showed that high yields of total phenols and total flavonoids could be obtained under the dark condition with a concentration of 1 mM phenylalanine and a concentration of 25 μM methyl jasmonate, indicating that during the plant cell suspension culture process, external environmental factors had a strong impact on the production and accumulation of flavonoids, such as anthocyanins and polyphenols, in plants.Apart from the choice of medium, the effects of certain external environmental factors, such as light, can also have important impacts on cell suspension culture. Andi et al.  comparedMoreover, since obtaining acylated anthocyanins by the biosynthetic pathway usually involves at least eleven genes, as described in previous reports , it is rAcylation of anthocyanins can also be performed in vitro through chemical approaches. The key to chemical acylation is the formation of an ester group since there are lots of -OH groups in anthocyanins, including those of anthocyanidins and glycosyl groups. Therefore, in chemical approaches, anthocyanin acylation is divided into the acylation of anthocyanidins and the acylation of anthocyanin glycosyls.Generally, the acylation of anthocyanins in plants mainly involves the partial or total esterification of -OH groups of anthocyanin glycosyl by various organic acids . As withRecent studies related to the chemical acylation of anthocyanins are summarized in In order to obtain acylated anthocyanins with high lipotropism, Cruz et al.  utilizedAlthough chemical acylation is a feasible approach to obtain acylated anthocyanins, the reactions are not region-selective, which may lead to unwanted functionalization of phenolic hydroxyl groups. External environmental factors, such as pH and ion concentration, affect the form and network of flavylium cations, hemiketals, quinonoid bases, and chalcones, which can lead to the transformation of anthocyanin structures and limit the progress of chemical acylation ,43.Enzymatic acylation of anthocyanins displays higher region-selectivity with high yield compared with chemical acylation and can be used to synthesize some special acylated anthocyanins with high stability and fat solubility under special conditions . EnzymatCruz et al.  reportedRibes nigrum L.), which was acylated with octanoic acid under the catalysis of Candida Antarctica lipase B It is difficult to find suitable plants rich in acylated anthocyanins, and the direct isolation and purification of single acylated anthocyanins from a large variety of anthocyanins in plants is a relatively cumbersome and costly process. Therefore, low valueable food by-products could be used as good sources for the isolation of anthocyanins by countercurrent chromatography (HSCCC) in subsequent studies, which would solve the problem of obtaining raw materials as well as achieve multiple effective uses of resources. In addition, later studies could develop alternative techniques, such as gene editing, gene transfer, and heterologous expression techniques, to construct several acylated anthocyanin bio-factories to obtain highly abundant acylated anthocyanins.(2)In vitro synthesis of acylated anthocyanins faces challenges of low reaction yields; therefore, subsequent research on the semi-biosynthetic acylation of anthocyanins could focus on investigating more suitable types of plant cells and optimizing the composition of media in order to obtain high yields of acylated anthocyanins. In addition, for the synthesis of acylated anthocyanins using chemical acylation or enzymatic acylation, research into the best reaction conditions, e.g., by optimizing the solvents used in reaction systems, and ways of increasing enzyme activity and developing the hybrid synthesis of acylated anthocyanins is also important.(3)Acylated anthocyanins usually face the problem of insufficient colour variety when they are used as food colourants. In order to tackle this challenge, on the one hand, innovative food colourants with a wide pH range, which contain acylated anthocyanins and other types of natural dyes, could be developed. When using acylated anthocyanins as food colourants, adjusting the pH values of food products could also be a useful way of enriching the colour of food colourants. On the other hand, natural anthocyanin pigments/dyes and phenolic co-pigments/co-dyes could be used to form non-covalent complexes to enrich the variety of food colourants. Since these non-covalent interactions allow dyes to interact with each other to produce co-pigmentation, the colours in flowers, berries, and foods made from them  could be stabilized and modulated.(4)Acylated anthocyanins have a variety of biological properties, such as anti-cancer, anti-inflammatory, anti-aging, anti-cardiovascular, and antioxidant properties, but the current studies on the biological activities of acylated anthocyanins are mostly at the cellular or preliminary mouse level. There are still not enough suitable primate models to validate the unique biological efficacies of acylated anthocyanins. Therefore, more animal models could be constructed, as well as relevant human clinical trials, to validate the biological activities of acylated anthocyanins in subsequent studies. In addition, safety evaluation models and methods for acylated anthocyanins need to be further refined.(5)Finally, when using acylated anthocyanins as indicators, a slight change in colour may not be observed by the naked eye, and thus their application in intelligent food packaging is challenging. More characterization methods could be introduced to reflect slight changes in colour. For example, traditional physical characteristics, such as CIELAB colour difference calculation, could be combined with molecular spectroscopy techniques, such as visible spectroscopy and near-infrared spectroscopy, to characterize changes in colour. New algorithms could also be developed to establish reliable correlation models to increase sensing accuracy.The recent advances in the mechanisms of acylation, including biosynthesis, semi-biosynthesis, and chemical and enzymatic acylation, and their applications in the food industry have been summarized in the current review. The acylation of anthocyanins through biosynthesis is performed in vivo by gene editing and regulation, while semi-biosynthesis and chemical and enzymatic acylation of anthocyanins are performed in vitro through the intervention of the external environment. Although these methods have some difficulties in directly obtaining pure acylated anthocyanins, they can be used to obtain relatively large quantities of acylated anthocyanins. With the further development of technologies, such as gene identification, gene editing, and gene expression in vitro, it will be possible to systematically elaborate the mechanism of biosynthesis of acylated anthocyanins. In addition, in recent years, more and more attention has been paid to the derivatization of polyphenols, and many studies have been conducted on the selection of enzymes and the optimization of reaction systems, such that enzymatic acylation has become an efficient method for the derivatization of more natural active substances, leading to the enrichment of certain types of active substances, such as polyphenols, and the provision of more beneficial active substances.


It is well known that the intestinal bacteria substantially affect physiological processes in many body organs. Especially, through a bidirectional communication called as gut-microbiota-brain axis, the gut microbiota deeply influences development and function of the nervous system. Hippocampus, as a part of medial temporal lobe, is known to be involved in cognition, emotion, and anxiety. Growing evidence indicates that the hippocampus is a target of the gut microbiota. We used a broad search linking the hippocampus with the gut microbiota and probiotics.All experimental studies and clinical trials published until end of 2021 were reviewed. Influence of the gut microbiota on the behavioral, electrophysiological, biochemical and histological aspects of the hippocampus were evaluated in this review.The effect of disrupted gut microbiota and probiotic supplements on the microbiota-hippocampus link is also considered. Studies show that a healthy gut microbiota is necessary for normal hippocampus dependent learning and memory and synaptic plasticity. The known current mechanisms are production and modulation of neurotrophins, neurotransmitters and receptors, regulation of intracellular molecular processes, normalizing the inflammatory/anti-inflammatory and oxidative/antioxidant factors, and histological stability of the hippocampus. Activity of the hippocampal neuronal circuits as well as behavioral functions of the hippocampus positively respond to different mixtures of probiotic bacteria.Growing evidence from animal researches indicate a close association between the hippocampus with the gut microbiota and probiotic bacteria as well. However, human studies and clinical trials verifying such a link are scant. Since the most of papers on this topic have been published over the past 3 years, intensive future research awaits. The gut microbiota refers to all the species of commensal cells comprising approximately 100 trillion microbes that inhabits the entire gastrointestinal tract. With over 1,000 species and 7,000 strains the microbiota is known as a dynamic ecosystem dominated by anaerobic bacteria, but also includes viruses and bacteriophages, protozoa, archaea, and fungi . The twoThe brain is an organ that is more susceptible to both internal and external environmental factors in adolescence and early adulthood. During this period, the neuronal architecture and function undergo rapid modulation to cope with environmental challenges . The gutHippocampus is the well-known area of brain involved in behavioral function. Particularly, it is a critical brain region associated with learning and memory and, in turn, is closely related to dementia and many other mental disorders such as depression and anxiety . For theFavorable effect of the gut microbiota on, at least, some cognitive brain function undoubtedly must be preceded through affecting the hippocampus. Although still scarce, however, evidence suggesting a direct link between the gut microbiota and hippocampal formation is increasing. In line, numerous studies have shown that the intestinal microbiota may affect hippocampal-related behaviors including learning and memory . The hipNormal function of the brain strongly depends on natural composition of the gut flora, known as “Eubiosis” . “DysbioLactobacilli and Bifidobacteria which are also normally found in healthy gut , trimethylamine N-oxide (TMAO) and amyloid beta (Aβ) in the gut microbiota could be affected by many treatments including antibiotics, germ free administration, diet, probiotics, prebiotics, and fecal microbiota transplantation . Probiotlthy gut . Hence, lthy gut . Probiotlthy gut . Evidenclthy gut . This syWe used a broad search string including “hippocampus AND gut microbiota” or “hippocampus AND probiotics.” All papers published until end of 2021 were considered in Web of Science and PubMed. Additional papers were obtained from references of identified papers. Registries for clinical trials were checked.Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) course of action were used in this study . The maiFirst-grade studies were those that directly link hippocampus to the gut microbiota and probiotics. The second-grade studies were those working on the factors from the others organs  or other parts of the CNS that affect the hippocampus.The reviewed studies were those focused on relationship of the gut microbiota and probiotic with the hippocampus. The findings consider hippocampus-dependent cognitive function, level of brain derived nerve factor (BDNF) in the hippocampus, the hippocampus synaptic plasticity, the histological changes in the hippocampus and the balance of anti-oxidant/oxidant and anti-inflammatory/inflammatory factors. Lactobacilli and Bifidobacteria were the most probiotic bacteria used in these studies. Streptococcus thermophilus, Akkermansia muciniphila, Porphyromonas gingivalis, Streptococcus faecalis, and Bacillus mesentericus were the other administered probiotics. In all cases the probiotic therapy improved the impaired learning and memory  in the hippocampus ; the small, non-coding RNAs which emerge as key post-transcriptional modulators of gene expression . They inThis review demonstrates that the intestinal bacteria has a powerful influence on the neuroinflammation so as to the gut microbiota has been proved to be an important neuroimmunomodulator . EvidencNeuroinflammation is associated with the cognitive impairment and memory decline, as the hippocampus is prone to develop alterations in synaptic transmission and plasticity during inflammation . WhereasThe intestinal microbiota is responsible for a significant production of SCFA  and the Despite aforementioned valuable roles of the gut microbiota, it may play injurious role under adverse conditions of the gut flora. TMAO, a gut microbiota metabolite, increases proinflammatory cytokine expression and microglia-mediated neuroinflammation. Also, structural bacterial components such as LPS provide low grade tonic stimulation of the innate immune system. Higher abundance of LPS is detectable in hippocampus of the people with Alzheimer’s disease . DysfuncCurrent research shows a close relationship between several brain disorders and changes in the gut microbiota ; promisiBecause this systematic review was devoted to the studies directly related to the hippocampus, very little data was available from human research and clinical trials, and so, at least in terms of cognitive function, we are just getting started.Therefore, human studies are encouraged to investigate the potential effects of the gut microbiota and probiotics on the hippocampus-related behaviors, particularly in brain diseases associated with cognitive disorders. Since majority of the papers considering relevancy of the hippocampus with the gut and probiotic bacteria have been published in the last 3 years, it promises a future with intensive research in this area.Overall, the production and modulation of neurotrophins, neurotransmitters and receptors, regulation of intracellular molecular processes, balance of inflammatory/anti-inflammatory and oxidative/antioxidant factors, and preservation of structural aspects of the hippocampus can be considered as important ways through them the gut microbiota affect the synaptic activity in the hippocampal neural circuits and hippocampus related behaviors. The supportive role of the probiotic bacteria, whether as supplementation for health or as therapeutic tool, is appreciated. However, the degree of efficacy of probiotics, optimal application paths, sex specificity, ages with the best most vulnerability to probiotics and the duration of treatment should be considered.The original contributions presented in this study are included in the article/MSa was the project leader of the review, designed the review, tabulated the review results, interpreted and discussed review findings, and did the screening, data abstraction, and quality appraisal. MSo conceived the review and prepared the tables and drawings. Both authors managed the review processes, summarized and described the review findings, and prepared the final version.


More than forty years after the first birth following in vitro fertilization (IVF), the success rates of IVF and of IVF-derived assisted reproduction techniques (ART) still remain relatively low. Interindividual differences between infertile couples and the nature of the problems underlying their infertility appear to be underestimated nowadays. Consequently, the molecular basis of each couple’s reproductive function and of its disturbances is needed to offer an individualized diagnostic and therapeutic approaches to each couple, instead of applying a standard or minimally adapted protocols to everybody. Interindividual differences include sperm and oocyte function and health status, early (preimplantation) embryonic development, the optimal window of uterine receptivity for the implanting embryo, the function of the corpus luteum as the main source of progesterone production during the first days of pregnancy, the timing of the subsequent luteoplacental shift in progesterone production, and aberrant reactions of the uterine immune cells to the implanting and recently implanted embryos. In this article, the molecular basis that underlies each of these abnormalities is reviewed and discussed, with the aim to design specific treatment options to be used for each of them. Despite the fact that the first successful in-vitro fertilization (IVF) attempt dates back more than 40 years ago , and manDespite the fact that, over the past 40 years, ART techniques have undergone continuous expansion, incorporating new methodological innovations, their basic steps remain the same . BrieflyOver the past few years, there have been more and more voices that campaign for the introduction of new strategies in order to improve the overall ART outcomes at the global level. Most of these voices agree on the need for abandoning the generalized use of routine or minimally adapted clinical and laboratory techniques in all infertile couples and adopting a personalized approach, based on molecular analysis of each individual case ,5,6.There are quite a few recent studies that deal with the different molecular mechanisms involved in the processes of gamete development and maturation, fertilization, pre-implantation embryo development, implantation and early post-implantation development. However, there is still a paucity of information about the links between the abnormalities of these mechanisms and a specific treatment to be used in the clinical setting.This short review summarizes the most significant recent findings related to the molecular events required for normal gamete development, fertilization, the early embryonic development, and uterine receptivity for the implanting embryo. These data can serve as a guide for future studies aimed at designing new diagnostic and therapeutic methods with which to improve the current efficacy of ART.After the introduction of micromanipulation techniques, namely intracytoplasmic sperm injection (ICSI) to assist fertilization ,8, many However, once the problem of oocyte activation had been resolved, a new serious obstacle, sperm DNA fragmentation, made its appearance. Sperm DNA fragmentation is caused by distinct mechanisms  and can Repair of both SSB and DSB can occur later, in the testicular germinal epithelium or in the zygote after fertilization . DiffereIn addition to sperm DNA fragmentation, mutations or abnormal expression of specific genes in male spermatogenic cells cause subsequent problems of embryo development, which is also the case for epigenetic disorders . Many ofAneuploid conception, resulting from chromosome segregation errors during the meiotic divisions in human female meiosis, is the main oocyte-derived cause of ART failure . Oocyte Analysis of genome-wide maps of recombination and chromosome segregation in human oocytes uncovered a new reverse chromosome segregation pattern concerning sister chromatid separation at meiosis I, influencing the elimination of aneuploid embryos and is responsible for the chromosomal drive against non-recombinant chromatids at meiosis II . MoreoveIt was shown recently that the propensity of individual chromosomes to meiotic errors is closely related to the number of crossovers (interchromatid recombination sites between chromatids of homologous chromosomes), forming synaptonemal complexes during the first meiotic prophase, chromosomes and oocytes with fewer crossovers being more susceptible to chromosome segregation errors ,32. FailEven though oocyte aneuploidies are responsible for most ART failures, especially in women of advanced age, other oocyte-borne ART failures are caused by specific gene variations, methylation pattern abnormalities, and inadequate gene expression levels .The age-related diminished ovarian reserve issue can hardly be resolved. However, it is rarely responsible for oocyte quality. The quality of oocytes is mainly dependent on the female age, and it can be ameliorated, to some extent, by medical treatments in most cases .Embryonic development is particularly sensitive to differential expression or mutation of numerous genes that control the pathways involved in cell cycle regulation, DNA repair and oocyte cytoplasmic maturation .Because of the relatively late onset of embryonic gene expression in humans and some other mammalian species see , the accPreimplantation embryo development encompasses the period between fertilization and embryo implantation in the uterus. Maternal-to-zygotic transition (MZT), during which transcripts of maternal genes are removed by degradation and the zygotic genome activation (ZGA) occurs , is a crZGA was shown recently to occur in two waves, both in mice  and in hIn addition to nucleologenesis, the early wave of human zygotic transcription also accounts for changes in the profile of small noncoding RNAs (sRNAs) that play important roles during MZT . AltogetIn view of the above consideration, preimplantation embryo development can be considered the main period during which the fate of the embryo unfolds, for better or worse. With respect to the timing of MZT, it can be subdivided into the following three main phases: pre-MZT, major ZGA, and accomplished MZT.Before the major ZGA, which occurs between the 4-cell and 8-cell stage of embryonic development in humans (see above), the developmental processes that take place in the embryonic cells are mainly under maternal control. During this period of transcriptional silence, development is driven by cytoplasmic factors, which largely consist of maternally deposited mRNAs, whose proper function requires strict temporal and spatial regulation . StudiesA recent study  analyzedEven though the timing of the major ZGA activation in different mammalian species, including humans, has been known since the 1980s ,44,45,46Transcriptomic data from human, mouse, and cow preimplantation development also show that the period of ZGA exhibits the highest level of mRNA alternative splicing and most of the exon skipping occurs in the genes responsible for DNA damage response . It was Progressive elimination of stored maternal mRNAs is required for MZT to be accomplished ,59. TranCytoplasmic polyadenylation-mediated translational control of maternal mRNAs directs ZGA and MZT in many mammalian and non-mammalian species . HoweverFuture work is needed to understand the mechanisms that regulate the reprogramming of the maternal sRNA profile, as well as confirming whether, when, and how miRNAs function during early embryonic development in humans. A systematic review and critical analysis of published data about molecular drivers of developmental arrest in the human preimplantation embryo have been published recently  and willUterine receptivity refers to a condition in which the uterus is suitable for embryo implantation, and it lasts for a limited time, which is defined as the implantation window. The concepts of uterine receptivity and implantation window were established in the 1960s, based on studies that employed the embryo transfer technique in different animal species . In humIn simple terms, the implantation window in all species studied so far is opened and closed by first messengers  whose action activates second messengers and downstream signal transduction pathways. Abnormalities at any of these levels can cause recurrent implantation failure (RIF) in humans, but the critical underlying molecular mechanisms still remain largely unknown .First messenger abnormalities responsible for embryo implantation failure have been most studied and represent a relatively easy target for treatment action, based on the concept of substitutive therapy. Estrogen and progesterone are the main first messengers responsible for the preparation of the uterus for embryo implantation . EstrogeSupraphysiological estrogen and suboptimal progesterone levels are common in ART attempts using different ovarian stimulation protocols and ovulation triggers, causing the condition known as luteal phase deficiency (LPD) . Low proIn addition to progesterone, growth hormone (GH) is another first messenger implicated in the acquisition of the optimal uterine receptivity in humans. This was first unequivocally demonstrated by a randomized controlled trial (RCT) that involved women with RIF after the transfer of embryos in an oocyte donation program, where oocytes were recovered from young donors not treated with GH . The RIFA number of other, non-hormonal ligands were also shown to affect uterine receptivity in animal models. These include leukemia inhibiting factor (LIF) , bone moBoth estradiol and progesterone, acting sequentially as the main drivers of uterine receptivity, bind to their nuclear receptors, acting as transcription factors through estradiol and progesterone response elements, respectively. The action of both hormones affects numerous post-receptor cell signaling elements, including growth factors, transcription factors, lipid mediators, cytokines, and cell cycle regulators involved in the initiation of pregnancy . In the Unlike estradiol and progesterone, GHR is located on the cell surface, and upon ligand binding, it activates the Janus kinase (JAK)-signal transducer and activator of the transcription (STAT) signaling pathway, in addition to activating the Src family kinase signaling pathway independent of JAK . InteresAmong the different factors responsible for pregnancy loss after implantation, diminished production of progesterone is particularly frequent after ART treatments, although it can also occur after natural conception . At the Despite the existence of a plethora of data related to the different molecular events that control fertilization, preimplantation embryo development and implantation, only few of them are at the origin of specific molecular-based therapies. This is partly due to the fact that most of these data stem from experiments carried out in animals, mainly rodents, while experiments in humans are restricted by ethical concerns. Unfortunately, with the exception of non-human primates, the mechanisms of early embryo formation, development and implantation show significant differences between experimental animals, and rodents in particular, on the one hand, and the human on the other hand. Hopefully, increasing knowledge of the abnormalities responsible for early developmental failures in the human will make it possible to design molecular treatment strategies, based on genome editing, in the future. However, despite enormous pressure by infertile couples whose problems cannot be solved by conventional methods, experts warn against the use of human germline genome editing, which may cause serious problems to the offspring; thus, all related techniques should be handled with care . NeverthAfter the advent of ICSI, many sperm abnormalities, leading to defective sperm movement, sperm-zona pellucida attachment, acrosome reaction and zona pellucida penetration, and sperm-oocyte fusion, can be easily overcome by ART. Hence, sperm DNA damage and deficiency of oocyte-activating factors remain the only serious sperm defects that still can cause ART failure in the ICSI era. Assisted oocyte activation (AOA), with the use of calcium ionophores , or mechSperm DNA damage can sometimes be mitigated by changing diet and lifestyle and by eliminating its primary cause, such as varicocele or infection, if it can be determined . HoweverThe other treatment strategy is based on the selection of spermatozoa with intact DNA by different laboratory techniques and is used in the context of ART. The techniques reported to be capable of selection against spermatozoa with damaged DNA, so as to facilitate the use of healthy spermatozoa for ART, include physiologic ICSI (PICSI), based on the capacity of intact spermatozoa to bind to hyaluronic acid, magnetic-activated cell sorting (MACS) , intracyAs is the case with male infertility, antioxidant treatment is also indicated in most cases of female infertility , especiaIn young women, antioxidants were mainly used in the following two clinical indications: polycystic ovary syndrome (PCOS) and endometriosis. In women with PCOS, treatment with resveratrol improved IVF outcomes, probably by changing the serum levels of some sex hormones and expression of VEGF and HIF1 genes involved in the angiogenesis pathway of granulosa cells . ImproveIn both young and older women, the main cell-signaling pathways involved in age-related or unrelated ovarian failure are those that control cell protection against oxidative stress . A singlHowever, the first substance proven to increase ongoing pregnancy and live birth rates in humans in a randomized controlled trial (RCT), published in 2005, was GH . SubsequThe exact mechanism of GH action in the human ovary still remains to be determined. Even though it is tempting to speculate that GH influences the signaling pathways that regulate intracelular redox balance, there is no experimental finding in favor of this hypothesis, and other possible mechanisms also have to be considered. For instance, GH co-treatment during ovarian stimulation for IVF in older women increases the density of receptors for FSH, LH and BMPR1B, as well as of its own receptors, in granulosa cells .The action of FSH and LH in this category of patients can also be enhanced by oral treatment with pentoxifylline, which supposedly acts by increasing the intracelular levels of cyclic adenosin monophosphate (cAMP), the second messenger that transduces the FSH-and LH-generated signal to downstream the elements of the pathway .As is the case with GH, melatonin is another multifaceted agent used in ART to improve oocyte and embryo quality. In 2008, a prospective study by Tamura et al.  showed tAs for impaired uterine receptivity in the context of ART, luteal phase deficiency (LPD)  and failUterine receptivity can also be disturbed by inadequate response of the endometrium to estrogen. In these cases, endometrium remains thin in the proliferative phase, despite the high doses of estrogen administered, and other adjunctive treatments, such as low-dose aspirin, pentoxifylline, tocopherol and vaginal sildenafil citrate , were evThese effects of G-CSF may be related to the fact that, as is the case with GHR  and LIFROther treatment methods suggested to improve endometrial thickness and receptivity, namely endometrial scratching and intrauterine injection of autologous platelet-rich plasma (PRP), intend to modulate the release of various autologous cytokines and growth factors to the uterine cavity. Endometrial scratching  was first proposed in 2003  and becaA hostile reaction of maternal endometrial natural killer (NK) cells to HLA antigens of the embryo, which actually represents an allograft carrying HLA-C antigens of both maternal and paternal origin, also represents a possible cause of RIF. Even though, in general, maternal NK cells play an important role in placentation and postimplantation embryonic development , the comVarious factors can cause human ART failures. These can be related both to sperm  and oocyte  function, as well as to abnormal interaction between the embryo to implant and the uterine environment . This means that sperm and oocyte quality must be checked before any ART attempt, and appropriate measures must be taken against each of them. Collateral factors that potentially cause ART failure  should also be taken into account. In case an abnormality is detected, appropriate treatment must be performed.


Gut microbiota is a microecosystem composed of various microorganisms. It plays an important role in human metabolism, and its metabolites affect different tissues and organs. Intestinal flora maintains the intestinal mucosal barrier and interacts with the immune system. The liver is closely linked to the intestine by the gut-liver axis. As the first organ that comes into contact with blood from the intestine, the liver will be deeply influenced by the gut microbiota and its metabolites, and the intestinal leakage and the imbalance of the flora are the trigger of the pathological reaction of the liver. In this paper, we discuss the role of gut microbiota and its metabolites in the pathogenesis and development of autoimmune liver diseases, metabolic liver disease such as non-alcoholic fatty liver disease, cirrhosisits and its complications, and liver cancer from the perspective of immune mechanism. And the recent progress in the treatment of these diseases was reviewed from the perspective of gut microbiota. The total number of genes is 100-400 times than that of human beings  expressed by intestinal flora activate pattern recognition receptors(PRRs) such as innate immune cells binding toll-like receptors (TLRs) to promote intestinal immune tolerance. When the flora is disturbed, MAMPs stimulate macrophages and dendritic cells to generate pro-inflammatory cytokines, leading to immune imbalance. Innate lymphoid cells(ILCs) are the lymphocyte population enriched in mucous membrane. It was found that the secretion of IFN-γ by ILCs can affect the composition and distribution of intestinal flora .Intestinal specific immunity is provided by the mucosal immune system. T cells and B cells in the mucosa and secretory immunoglobulin A(sIgA) secreted into the intestinal lumen jointly complete the local intestinal mucosal immune function . Intestiin vivo  and lipid teichoic acid(LTA) are the membranes of the cytoderm of Gram-negative bacteria and Gram-positive bacteria in the intestinal tract, respectively. Under normal circumstances, intestines are high in LPS and LTA. If LPS and LTA continuously stimulate the innate immune system of the intestinal tract to produce inflammatory response, it will cause very serious immune diseases . PeptidoGut microbiota can produce metabolites, such as short-chain fatty acids (SCFAs), trimethylamine oxide (TMAO), bile acids(BAs), tryptophan and its metabolites, pyruvate and lactic acid, through food degradation, biotransformation and secretion. Dietary fiber is fermented through gut microbiota to produce SCFAs. 90%~95% of SCFAs in intestinal tract are acetic acid, butyric acid, propionic acid . As the Since Marshall proposed the concept of gut-liver axis in 1998, intestinal relations with liver disease more and more get the attention of people . The gutvia Treg cells and activates the nuclear factor-κB (NF-κB) by TLR and Nod-like receptor(NLR) via the pathogen-associated molecular pattern(PAMP). The study confirmed that the expression of TJ-related proteins occludin and Claudin-1 was significantly reduced, leading to increased intestinal permeability and endotoxemia  or activated by the adaptive immune system otoxemia . The resotoxemia . On the otoxemia Figure 2otoxemia . The prootoxemia . The secotoxemia . In the otoxemia . Damage The synthesis and secretion of BAs in liver and their reabsorption in the intestine form the gut-liver cycle. BAs are produced by cholesterol in liver and metabolized in gut by gut microbiota. About 90% to 95% of BAs are absorbed at the far end of the distal ileum and then transported to the liver, where they recombine with taurine (in mice) or glycine (in humans) to form bile salts. About 5% to 10% of BAs are degraded and biotransformed by microorganisms mainly in the intestinal tract, and some of them are excreted by feces. The transformation of BAs in the intestinal tract is mainly accomplished by Bacteroidetes, Eulobacter and Clostridium in the intestinal anaerobe. Taurine and glycine are decombined with bile salts by the action of bile saline hydrolysis enzyme to form separate free BAs . The modAutoimmune hepatitis(AIH) is an abnormal immune reactivity-mediated intrahepatic inflammatory disease targeting hepatocytes, characterized by varying levels of elevated serum transaminase, positive characteristic autoantibodies, hypergammaglobulinemia, and characteristic changes in liver histology, and usually responds well to immunosuppressive therapy . The dis+ AIH models, but not in wild-type mice, suggesting that genetic factors may induce changes in the mouse microbiota and are related to AIH susceptibility . How to avoid or minimize the side effects caused by long-term treatment is the focus of clinicians. Probiotics can not only restore the composition of the intestinal flora, but also have the function of regulating the immune system. Probiotics are safe and effective in alleviating AIDs and are expected to be adjunctive or even alternative therapies. Some researchers gave AIH mice intragastric administration of compound probiotics, and found that it could inhibit the infiltration of inflammatory cells in the liver, reduce Th17 and Th1 cells and serum transaminase, and reduce AIH . MoreovePrimary biliary cholangitis(PBC) is a chronic intrahepatic cholestasis with the most common clinical manifestations of fatigue and itchy skin, which can lead to liver failure or even death . PBC feaGut microbiota can affect the pathogenesis and progression of PBC at multiple levels. Some scholars have found that fecal microbiota is associated with fibrosis and cirrhosis of PBC, and a decrease in alpha diversity and an increase in Weisella in advanced fibrosis community . The micUrsodeoxycholic acid(UDCA), a first-line treatment for PBC, is efficient in about two-thirds of patients with early PBC , 80. Theas increased only in the inferior remission(IR) group . AccordiPrimary Sclerosing Cholangitis (PSC) is characterized by idiopathic intrahepatic and/or extrahepatic bile duct diffuse inflammation and fibrosis, leading to multifocal bile duct stenosis and chronic cholestasis . The patAt present, there have been many researches on the changes of intestinal flora in PSC patients, and the specific changes of the flora have also been analyzed more clearly. Study  found anCholangiocytes are specialized cells arranged in the hepatic duct network. Cholangiocytes not only play roles in bile modification and transport,but also are capable of sensing endogenous and exogenous molecules, including PAMPs . This waFurthermore, intestinal flora may also contribute to PSC/PSC-IBD nosogenesis through co-metabolism of synthetic compounds or host-produced molecules. For example, the BAs mentioned above. BAs acts as a key signaling molecule through interactions with several receptors, including the TGR5 and the FXR. These receptors play multiple roles the liver and gut . TGR5 agAt present, most clinical studies or animal experiments focus on exploring the relationship between PSC and intestinal flora dysbiosis, as well as the changes of intestinal flora in PSC patients or animal models. By far, most clinical studies targeting at gut microbiota were small-sample trials, multi-center and large-sample studies are urgently needed to provide higher-level evidence. Several small-sample clinical studies –106 withNonalcoholic fatty liver disease (NAFLD) is a common clinical liver disease, which refers to the chronic metabolic diseases caused by diffuse fatty infiltration of hepatocytes caused by other factors except excessive drinking, including non-alcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH), and related liver fibrosis. The disease can develop into liver cirrhosis and even hepatocellular carcinoma (HCC) without treatment . The occBAs are reabsorbed into the portal vein, where most molecules are captured by the liver, some remain in the bloodstream, where they act as signaling molecules through receptors such as TGR5 and FXR , 116. AcIn addition to affecting the metabolism of BAs and choline, the gut microbiota also produces metabolites, such as SCFA and endotoxins (LPS), which are also related to the pathological development of NAFLD. Studies have shown that SCFAs mediate modulation of intestinal flora and host inflammatory responses, for example, butyrate activates regulatory T cells in the gut, which in turn inhibit T cells and Th17 cells, thereby reducing pro-inflammatory signaling pathways , 122. DeGiven the critical role of intestinal flora biota dysplasia in NAFLD pathogenesis, significant progress has been made to explore ways in which gut microbiota biota biodistribution and elucidated metabolites can be corrected. These methods include the use of probiotics, prebiotics and synthetic bacteria, as well as the use of fecal microbiota transplantation(FMT).These intervention modes have been widely studied in clinical trials and animal models and achieved good results. Various probiotics were tested in NAFLD animal models. And the results obtained are promising . LactobaThe intestinal flora is involved in NAFLD. Different stages of NAFLD have different characteristics with intestinal flora .. ChangeCirrhosis is the terminal stage during the development of various chronic liver diseases and is widely prevalent worldwide. It is caused by heavy alcohol consumption, obesity, NAFLD, viral infections, cholestatic diseases, AIDs, and copper or iron overload . As the On the one hand, cirrhosis can cause intestinal microbiota changes , on the Hepatic encephalopathy(HE) is a syndrome caused by chronic or acute liver failure, decompensated cirrhosis, or various abnormal portal veno-systemic circulation shunt. HE manifests itself in a wide range of neuropsychiatric disorders, from mild cognitive impairment to significant disorientation, delirium and coma . Some guAscites is one of the common complications in the late stage of liver disease and an important marker of disease progression. The case fatality rate of 1 year after the occurrence of ascites is about 15%, and that of 5 years is up to 44%-85% , 164. GoSpontaneous bacterial perionitis (SBP) is a peritoneal infection caused by pathogenic bacteria passing through the intestinal, blood or lymphatic system in the absence of definite intraperitoneal disease based on cirrhosis, and is a common complication and cause of death in patients with end-stage liver diseases. The most common symptoms of SBP are chills, fever and abdominal pain. The most common infection source of SBP is Gram-negative bacteria, and intestinal bacterial translocation plays a crucial part in the occurrence and development of SBP. SBP occurs when bacteria normally living in the gut enter the abdominal cavity and the ascites become infected. This occurs in advanced liver disease because of a weakened immune system response and changes in the bacterial environment in the gut . EnhanceConventional treatment of cirrhosis focuses on the treatment of causes and complications, and in some cases may require a cases liver transplantation. Correcting gut microbiome imbalance can alter the progression of cirrhosis . AntibioLiver cancer generally progresses from chronic liver diseases(CLD), and most types of liver diseases have a potential risk of developing liver cancer. Alterations in intestinal permeability and intestinal microbiota composition are prominent features of all sorts of end-stage CLDs, and also appear in the initial stage of some CLDs. Hence the gut microbiota can affect the progression of CLDs at different periods and may give contribution to the progress of liver cancer in all of these periods. As mentioned earlier, the gut-liver axis is the way in which the intestinal flora affects the liver.Recently, increasing evidence suggested that disruption of the gut-liver axis contributed to the development of most kinds of CLDs, involving liver cirrhosis and liver cancer . The gutMicrobiota dysbiosis alters the metabolic pathways of the gut microbiota , therebyThere are a large number of immune cells in the liver, and there was experimental evidence that innate or adaptive immune responses can promote or inhibit the progress of liver cancer . Part ofSince gut flora has been found to be a vital role in promoting the progress of CLDs and liver cancer in animal experiments, it is easy to consider the intestinal flora as an important target for liver cancer treatment . VariousAbove we have concluded how the gut microbiota affects liver cancer through leaky gut and dysbiosis, and also introduced potential treatments aiming at gut microbiota for liver cancer. Clinical studies, especially randomized controlled studies, and the specific pharmacological mechanism are still lack of investment, so further exploration is urgently needed.Gut microbiota is usually considered to be closely related to metabolic diseases. Since our human body is an organic whole, exploring this vital symbiotic ecosystem will surely lead us to further comprehension to various diseases. At the same time, the intestinal flora can also be regarded as a corresponding therapeutic target, providing another perspective for the development of clinical pharmacy. Therefore, it is of great significance to clarify the role of the intestinal flora in diseases of different systems. In this article, we reviewed the role of the gut microbiota in various liver diseases and interventions that target at it for therapy. In general, the liver and the intestine have a close connection through the gut-liver axis. The destruction of the intestinal barrier and the imbalance of the gut microbiota are the initiating factors that cause hepatic lesions. In different kinds or stages of hepatopathy, the composition of the gut microbiota is disparate. LPS, SCFAs, BA, and other bacterial metabolites are the main signals inducing immune responses in liver tissue. The TLR family plays a key role in mediating these signals to produce liver inflammation. In terms of treatment, antibiotics, probiotics, prebiotics, synbiotics, and fecal microbiota transplantation are the main therapeutic methods targeting at gut microbiota, and their effectiveness has been confirmed in animal experiments and some small clinical studies.However, current researches on the gut microbiota and liver diseases also face the following challenges. First of all, most of these researches are animal experiments, which aim to explain the potential correlation between gut microbiota and liver diseases, and few of them can form an exact and coherent pathological mechanism. Therefore, more in-depth human researches are urgently needed to verify these potential mechanisms. Secondly, the intestinal flora is susceptible to genetic, environmental, behavioral and other factors, and further multi-racial, long-term, multicenter studies and better matching controls are needed to explore the association between the intestinal flora and specific diseases.The diversity of gut flora and the differences between individuals make it important to look for core flora that really works. With the development of sequencing technology and the revolution in metagenomics, enterotype have been proposed to characterize the intestinal flora . ThroughAll the authors participated with suggestions and the development of this manuscript. LW and ZC participated in drafting the article and/or revising it critically for important intellectual content, and also created the figures and tables. WL and LZ participated in the study conception, critical revision of the article, and supervision. JL has made great contributions to the revision process of the article.National Key Research and Development Program of China (No. 2018YFC1705700).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.