Prebiotic Effects of Chinese Herbal Polysaccharides on NAFLD Amelioration: The Preclinical Progress

Abstract

Nonalcoholic fatty liver disease (NAFLD) is caused by fatty degeneration of liver cells, and there are currently no effective treatments. Numerous investigations have demonstrated that Chinese herbal medicines (CHMs) are effective against NAFLD. Polysaccharides (PS), the major components of most CHM, are primarily taken orally to be degraded and fermented by gut microbiota, which makes them a promising multivalent and multifunctional prebiotic candidate for NAFLD. In this review, the experimental evidence to prevent and treat NAFLD using the unique prebiotic effects of PS isolated from CHM are summarized to discuss additional treatment options for NAFLD.

Keywords

nonalcoholic fatty liver disease traditional Chinese medicines herbal polysaccharides gut microbiota dysbiosis bioactivity

Introduction

Nonalcoholic fatty liver disease (NAFLD) refers to diseases that are caused by the fatty degeneration of liver cells, which includes nonalcoholic fatty liver (NAFL, simple steatosis), and nonalcoholic fatty steatohepatitis (NASH).¹ NAFL is defined as an abnormal accumulation of triglyceride (TG) in the liver but without inflammation or liver cell damage. When liver lobule inflammation and hepatocellular damage occur in the liver of chronic patients, NAFLD progresses to NASH, which could lead to liver fibrosis, liver cirrhosis, and hepatocellular carcinoma.² In addition, NAFLD might increase the risk of developing diabetes and cardiovascular disease.³ The incidence of NAFLD has steadily increased over the last few decades, and it is now estimated to be 25% worldwide.⁴ Li et al discovered a 30% prevalence of NAFLD in Asia in their recent systematic review and meta-analysis, which included 237 studies from 1999 to 2019.⁵ Another meta-analysis by Zhou et al of 392 studies revealed a 29% prevalence of NAFLD in China.⁶ The increase in the prevalence of NAFLD in Asia is primarily due to dietary and lifestyle changes.^7,8 Currently, NAFLD is considered to be a liver manifestation of metabolic disorders and obesity, hyperlipidemia, and insulin resistance (IR) have been identified as risk factors for disease development,^9–11 but the exact pathogenesis mechanism is not understood.¹² Of note, numerous investigations have linked dysbiosis to the occurrence and progression of NAFLD.^13,14

The gut microbiota in the human gastrointestinal tract consists of trillions of microorganisms, which includes bacteria, archaea, viruses, and eukaryotic microorganisms, the majority of which are bacteria. Over 90% of the human intestinal flora is composed of 5 phyla: Firmicutes (79.4%) (Ruminococcus, Clostridium, and Eubacteria), Bacteroidetes (16.9%) (Porphyromonas, Prevotella), Actinobacteria (2.5%) (Bifidobacterium), Proteobacteria (1%), and Verrumicrobia (0.1%).¹⁵ Bacteria, such as Bacteroides, Bifidobacterium, Faecalibacterium sp., and Enterobacter sp. are involved in the synthesis of short-chain fatty acids (SCFAs), such as butyrate, propionate, and acetate, and Bacteroides thetaiotaomicron helps to hydrolyze lipids.¹⁶ In a healthy state, the host and intestinal flora work cooperatively to maintain a relative balance, which is known as normobiosis; however, in pathological conditions, the ratio of symbiotic to pathogenic bacteria becomes abnormal, which results in dysbiosis, a structural and functional disorder of microbiota.¹⁷ Therefore, dysbiosis has emerged as a new target for NAFLD therapy. In addition, clinical studies have demonstrated that Chinese herbal medicines (CHMs) have a beneficial effect on NAFLD.¹⁸ Most of the CHM substances are derived from herbal plants and polysaccharides (PS) are one of their major components. These PS have gained considerable attention recently due to their significant biological activities, including antitumor, antioxidant, antidiabetic, radiation-protective, antiviral, hypolipidemic, and immunomodulatory properties. Most CHMs are taken orally, which causes them to be fully exposed to the gut microbiota and affect human health by interacting with the microbiota.^19,20 In this report, the research progress in the prevention and treatment of NAFLD by regulating the microbiota with PS, which are a key component of CHMs and could provide direction and foundations to explore additional treatment options for NAFLD is summarized.²¹

Chinese Herbal PS and Prebiotics

Chinese Herbal PSs

Most of the ingredients in CHMs are derived from herbal plants and PS are major components, and their biosynthesis is influenced by various environmental factors. Recently, various biological activities of Chinese herbal PS, such as antitumor, anti-oxidation, hypolipidemic, and immunomodulatory have been investigated and have received increased attention.^22,23 These bioactive PS are mostly isolated via hot water leaching, which employs a similar compatibility principle to dissolve the polar polymer compound PS found in water and other polar solvents. Recently, various novel isolation and purification techniques have been developed, which include enzyme-assisted extraction, microwave-assisted extraction, ultrasonic-assisted extraction, and supercritical fluid extraction.^23,24 The monosaccharide composition of Chinese herbal PS usually includes arabinose, fructose, fucose, galactose, glucose, mannose, rhamnose, ribose, and xylose in different molar ratios.²² In general, herbal PS cannot be digested in the upper digestive tract but can enter the distal intestine to be degraded and fermented by the gut microbiota.^25,26 More importantly, increasing evidence has demonstrated that the interaction between the processing gradients of herbal PS with the gut microbiota could benefit human health via their unique prebiotic effects.²⁷

Prebiotics Effects of Herbal PS

Recently, research into prebiotic-mediated dysbiosis interventions has accelerated exponentially. The term prebiotic, which was introduced by Gibson and Roberfroid in 1995, generally refers to nondigestible food ingredients that could promote the growth, or activity, or both of certain bacterial strains in the gut, which results in health benefits for the host.^28,29 The representative prebiotics mainly includes oligosaccharides, such as fructooligosaccharides, galactooligosaccharides, isomaltooligosaccharides, xylooligosaccharides, lactulose, and soy oligosaccharides, and PS, such as inulin, cellulose, hemicellulose, pectin, and resistant starch.³⁰ Chinese herbal PS are taken orally. In addition, they need to be fermented by the gut microbiota for metabolism, because they are not digestible in the upper digestive tract. Combined with their PS or oligosaccharide nature and the degradation products, these properties collectively make PS that are derived from CHMs theoretically fit the definition of a prebiotic quite well.³¹

In addition, the prebiotic effects of Chinese herbal PS have been gradually revealed. For example, Zou et al extracted PS from Codonopsis tangshen by fractionation (CTPN) on a DEAE-Sepharose column and confirmed that they were β-(2,1)-linked inulin-type fructans with nonreducing terminal glucose and a degree of polymerization (DP) of 25.2. Its prebiotic effects were further confirmed by the stimulation of Lactobacillus growth in vitro.³² In another study, Pang et al obtained the inulin-type fructan (PGF) by DEAE anion exchange chromatography from Platycodon grandiflorus (Ji Geng in Chinese). Methanolysis, methylation, and nuclear magnetic resonance (NMR) results revealed that PGF was a β-(2-1) linked fructan, with terminal glucose and a DP of 2-10. In addition, they proved that PGF had beneficial prebiotic activity by promoting the growth of 6 Lactobacillus sp.³³ The pathogenic effects of dysbiosis in NAFLD and the prebiotics intervention with Chinese herbal PS will be discussed in detail in the following sections (Table 1).

Table 1.

Representative Prebiotic Effects of Chinese Herbal PS on Ameliorating Experimental NAFLD.

Herbal PS	Extraction methods and chemical futures	Animal model	Prebiotic effects	Metabolism ameliorating in liver	Anti-inflammation in liver	Ref.
G lucidum (LingZhi)	High molecular weight PS (>300 kDa) isolated from the water extract of G lucidum (WEGL) (>300 kDa)	HFD mice	Decrease Firmicutes-to-Bacteroidetes ratios and endotoxin-producing Proteobacteria levels; FMT confirmed	Reduce liver weight gain, fat accumulation, and IR; FMT confirmed	Reduce mRNA expression of inflammatory cytokines, macrophage infiltration and TLR4 signaling; FMT confirmed	³⁴
	PS extracted from the sporoderm-broken spores of G lucidum (BSGLP)	HFD mice	Decrease Firmicutes-to-Bacteroidetes ratios; increase SCFAs production; FMT confirmed;	Inhibit fat accumulation; FMT confirmed	Inhibit TLR4/MyD88/NF-κB signaling activation; FMT confirmed	³⁵
H sinensis (DongChongXiaCao)	Fraction H1 containing high molecular weight PS	HFD mice	Increase P goldsteinii levels; decrease gut permeability	Inhibit fat accumulation and IR; FMT confirmed	Inhibit liver cell hypertrophy; FMT confirmed	³⁶
A. mongholicus (HuangQi)	APS prepared by water extraction, deproteinization and dialysis; monosaccharides compositions include rhamnose, arabinose, xylose, glucose, and galactose	HFD mice	Increase Bacteroidetes phylum abundance and inhibits Actinobacteria, Firmicutes, and Proteobacteria phyla	Reduce liver weight gain, hepatic steatosis, and IR	NA	³⁷
P kingianum (YuZhu)	PS (178.6 kDa) and PS PSF (134.7 kDa) prepared by water extraction, deproteinization and dialysis; monosaccharides composition include mannose, galacturonic acid, galactose, and fucose	HFD rats	Decrease the abundances of Bacteroidetes and Proteobacteria, and increase that of Firmicutes; increase the abundance of SCFAs producing bacteria, such as Bifidobacterium and Streptococcus; reduce LPS levels in levels in the hepatic portal vein and increase intestinal expression of ZO-1 and occludin	Reduce liver weight gain and hepatic lipid deposition; reduce TG, TC, AST, ALT, and PPARγ levels in the liver; inhibit IR	Inhibit TLR4/NF-κB pathway and PPARγ expression; decreased the levels of IL-1β and increased the levels of IL-10 in the hepatic tissues	^38,39
O japonicus (MaiDong)	MDG-1, the water-soluble β-d-fructan extracted from the roots of O japonicus	HFD mice	Decrease Firmicutes:Bacteroidetes ratio; increase the abundance of SCFAs producing bacteria, such as Streptococcus and Ruminiclostrium	Reduce liver weight gain and hepatic lipid deposition; reduce AST and ALT levels; active hepatic AMPK pathway	Reduce the expression of inflammatory cytokines	^40,41
Inulin (JuFen)	Natural prebiotic found in herbs	HFD mice	Up-regulate the abundances of Akkermansia and Bifidobacterium; downregulate the abundances of Blautia, Ileibacterium and Firmicutes/Bacteroidetes ratio; increase intestinal SCFAs	Reduce liver weight gains; reduce TG, TC, AST, and ALT levels in the liver;	Inhibit proinflammatory cytokines expression; inhibit the proportion of hepatic TLR4 + Mψs; inhibit the NF-κB mediated NLRP3 inflammasome activation	⁴²
P cocos (BaiZhu)	WIP isolated and identified from the sclerotium of P cocos; the (1-3)-β-D-glucan (4486 kDa) identified by NMR and SEC-RI-MALLS analysis	ob/ob mice	Increase butyrate-producing bacteria Lachnospiracea and Clostridium; elevate the level of butyrate in gut; improve the gut mucosal integrity; activate the intestinal PPAR-γ pathway	Improve glucose and lipid metabolism and alleviated hepatic steatosis; FMT confirmed	Reduce expression of proinflammatory cytokines; inhibit TLR4 signaling	⁴³

Abbreviations: NAFLD, nonalcoholic fatty liver disease; FMT, fecal microbiota transplantation; IR, insulin resistance; TLR, toll-like receptor.

Dysbiosis and NAFLD

The Correlation Between Dysbiosis and NAFLD

Studies have shown that dysbiosis has been linked to the pathogenesis of NAFLD.^44,45 The most typical dysbiosis changes include a decrease in microbiota diversity.⁴⁶ However, due to the complexity of NAFLD and the heterogeneity of the study subjects, clinical studies make it difficult to determine whether dysbiosis is one of the causes of NAFLD or just one of the associated abnormalities. In contrast, rigorously controlled intervention experiments that use mouse and rat models could more conclusively demonstrate the direct pathogenic effect of dysbiosis on NAFLD.

For instance, several fecal microbiota transplantation (FMT) studies have been conducted based on the dysbiosis that was established in animal models induced by one of the NAFLD risk factors, the high-fat diet (HFD).⁴⁷ Le Roy et al found that the FMT transferred from the NAFLD mice model into sterile mice caused the receiver to show the disease characteristics of NAFLD, which included hyperlipidemia and hepatocyte steatosis.⁴⁸ Soderborg et al demonstrated that germ-free mice that received FMT from mice born to obese mothers (rather than from babies of lean mice) developed NAFLD-like hepatic changes and periportal inflammation.⁴⁹ In addition, experiments that involved the transfer of FMTs from NAFLD patients vilified the role of dysbiosis in the pathogenesis of NAFLD. Chiu et al found that HFD germ-free mice that received FMTs from NASH patients developed increased epididymal fat weight, hepatic steatosis, inflammation and multifocal necrosis, elevated levels of ALT, AST, endotoxin, IL-6 and Mcp1, and increased toll-like receptor 2 (TLR2), TLR4, TNF-alpha, Mcp1, and PPAR-gamma mRNA expression.⁵⁰ Hoyles et al discovered that FMT from NAFLD patients caused hepatic steatosis and typical NAFLD-related dysbiosis in germ-free mice.⁵¹ These findings illustrated that dysbiosis is associated with, and contributes to, the development of NAFLD.

The Underlying Dysbiosis Mechanism in NAFLD Pathogenesis via the Gut–Liver Axis

The intestine and liver develop from the embryonic foregut. Metabolites in the intestine are absorbed into the portal vein via the superior and inferior mesenteric vein, which provide 70% of the blood supply to the liver. Therefore, the 2-way relationship formed by the close anatomy and physiology of the intestine and liver via the biliary tract, portal vein, and systemic circulation is called the gut–liver axis.⁵² As a critical component of the gut–liver axis, microbiota and its bacterial components and metabolites, enter the liver portal vein.⁵³ Therefore, dysbiosis has a significant role in a variety of liver diseases, which include NAFLD via the gut–liver axis.^13,14

Studies revealed that dysbiosis could be implicated in the pathogenesis of NAFLD in the following ways: (1) increasing nutrient absorption and fat accumulation. Dysbiosis causes indigestible carbohydrates to be fermented into an absorbable form; therefore, promoting nutrient absorption and energy intake when impairing fat degradation and conversion, which results in the accumulation of TG in the liver;^54,55 and (2) inhibiting the production of bile acid and SCFAs: Bile acid enterohepatic circulation is critical to maintaining the balance of lipid and carbohydrate metabolism.⁵⁶ Dysbiosis inhibits bile acid synthesis, metabolism, and reabsorption; therefore, accelerating fat synthesis and inducing NAFLD.^57,58 SCFAs, which are primarily produced by beneficial bacteria in the microbiota via the degradation and fermentation of food carbohydrates, reduce inflammation and lipid deposition in the liver, promote insulin release, and maintain the number of β cells.^59,60 By reducing SCFAs production, dysbiosis might contribute to the pathogenesis of NAFLD;⁶¹ (3) Increasing intestinal permeability. Dysbiosis increases intestinal permeability by downregulating the expression of intestinal tight junction proteins, such as ZO-1 and occludin, which allows a large number of bacteria and derivatives to enter the liver via the portal vein and become significant phenotypes and pathogenic factors of NAFLD;^62,63 (4) Promoting lipopolysaccharide (LPS) induced hepatic inflammation and IR; LPS that is produced by microbiota of dysbiosis enters the liver via the damaged intestine and the gut–liver axis. By binding to the TLR4 on the cell surface, LPS stimulates the production of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin-1-beta (IL-1β), interleukin-6 (IL-6) in hepatocytes and Kupffer cells and disrupts the insulin signaling pathway, which results in IR and NAFLD.^35,64

The Prebiotic Effects of Chinese Herbal PS in Experimental NAFLD

Ganoderma lucidum (LingZhi in Chinese): G lucidum is a curative mushroom in CHMs with putative metabolism-regulating activities. Chang et al found that the water extraction of G lucidum mycelium (WEGL) reduced liver weight gain, fat accumulation, mRNA expression of inflammatory cytokines, macrophage infiltration, TLR4 signaling, and IR in the hepatic tissues of HFD mice. In addition, WEGL decreases Firmicutes:Bacteroidetes ratios and endotoxin-producing Proteobacteria levels. In addition, the high molecular weight PS (>300 kDa) in WEGL exhibited comparable anti-NAFLD and prebiotic activities.³⁴ Recently, Sang et al showed that PS of the sporoderm-broken spores of G lucidum (BSGLP) inhibited hepatic fat accumulation, inflammation, and TLR4/MyD88/NF-κB signaling activation in HFD mice. In addition, BSGLP improved HFD caused gut dysbiosis, maintained gut barrier, and increased SCFA production, which was confirmed by an FMT study.⁶⁵ These studies indicated that the use of PS from G lucidum as prebiotics could prevent and treat NAFLD.

Hirsutella sinensis (DongChongXiaCao in Chinese): Ophiocordyceps sinensis and its anamorph H sinensis have been used in CHMs to modulate immunity for centuries. Wu et al revealed H sinensis mycelium (HSM) and the H1 fraction that contained high molecular weight PS increased Parabacteroides goldsteinii levels and reduced gut permeability in HFD mice. Specifically, HSM or H1-alleviated NAFLD and NASH-related disorders, such as hepatic cellular hypertrophy, lipid accumulation, and IR, as confirmed by FMT, which suggests that HSM and H1 induced P goldsteinii increases might target specific NAFLD-related hepatic metabolic and inflammation pathways.³⁶

Polygonum multiflorum (HeShouWu in Chinese): P multiflorum, which is officially listed in the Chinese Pharmacopoeia, is a well-known CHM herb that is used to treat various medical conditions, which include liver injury and diabetes. The lipid-lowering activity of P multiflorum has been investigated in animal models of NAFLD.⁶⁶ Gu et al found that P multiflorum relieved the liver steatosis by downregulating LDL-C and upregulating HDL-C in IR rats. In addition, they demonstrated that P multiflorum reduced Firmicutes:Bacteroidetes sp. ratios in the gut.⁶⁷ Another study from the same group revealed that PS from P multiflorum significantly inhibited IR and effectively reversed the increased Firmicutes:Bacteroides ratio in HSD rats, which resulted in a significantly decreased abundance of Proteobacteria, and elevated SCFAs and their downstream molecules.⁶⁸

Astragalus mongholicus (HuangQi in Chinese): According to various studies, PS is a significant bioactive component of A membranaceus with a range of health benefits.⁶⁹ Hong et al found that Astragalus PS (APS) inhibited liver weight gain, hepatic steatosis, and IR in HFD mice. Metagenomic and metabolomic results revealed that APS reversed the disorders of 188 species mainly from Bacteroidetes, Actinobacteria, Firmicutes, and Proteobacteria phyla, and 36 related metabolites in HFD mice, which indicated the potential prebiotic benefits of APS in NAFLD.³⁷

Polygonatum kingianum (YuZhu in Chinese): P kingianum is a CHM herb widely used for diabetes and hyperlipidemia. Yan et al found that total polysaccharides (PSPK) prevented IR development in HFD rats. In addition, PSPK increased the abundances of Bacteroidetes and Proteobacteria and the contents of SCFAs and decreased that of Firmicutes.³⁸ Wen et al found that PS and polysaccharide fractions (PSF) from P kingianum prevented liver weight gains, reduced hepatic lipid deposition, TLR4/NF-κB immune responses, and PPARγ expression significantly. In addition, PS and PSF restored the relative abundance of microbiota to normal levels and increased SCFAs production in HFD rats, which suggested that PS prevents liver glucose and lipid metabolic disorders by increasing the relative proportion of SCFA-producing gut microbiota, such as Bifidobacterium and Streptococcus.³⁹

Ophiopogon japonicus (MaiDong in Chinese): The PS found in abundance in O japonicus (Chinese Pharmacopoeia Commission, 2015) might be responsible for its therapeutic properties.⁷⁰ MDG-1 is a β-d-fructan from the water extraction of the O japonicus roots. Shi et al revealed that in HFD mice, MDG-1 decreased the Firmicutes:Bacteroidetes ratios and helped to promote weight loss and effective energy metabolism.⁴⁰ Wang et al revealed that MDG-1 significantly reduced liver weight gains, hepatic lipid deposition, AST and ALT levels, the expression of inflammatory cytokines and activated the hepatic AMP-activated protein kinase (AMPK) pathway in HFD mice. In addition, MDG-1 reduced Firmicutes:Bacteroidetes ratios and increased the abundance of SCFAs producing bacteria, such as Streptococcus and Ruminiclostrium.⁴¹

Inulin (JuFen in Chinese): As a typical prebiotic in herbs, inulin helps to rebuild gut normobiosis. Yang et al found that inulin ameliorated the chronic ethanol (EtOH) intake induced liver inflammation, which includes hepatic cytokine expression and TLR4⁺ hepatic macrophages (Mψs) proportion. 16S rRNA sequencing indicated that inulin restored anti-inflammatory bacteria, such as Allobaculum, Lactobacillus, and Lactococcus and decreased inflammatory microbiota strains, such as Parasutterella.⁷¹ Their in vitro results demonstrated that inulin improved liver inflammation via a SCFAs mediated polarization switch from M1 to M2 macrophages.⁷² In NAFLD mice, researchers from the same group discovered that inulin restored the abnormal hepatic indicators that were induced by HFD, such as significant increases in liver weight, ALT and AST, TG, total cholesterol, and hepatic inflammatory cytokines expression. In addition, the relative proportion of hepatic TLR4⁺ macrophages and the expression of NFκB, nod-like receptor protein 3, apoptosis-associated speck-like protein, and caspase-1 in liver were inhibited with inulin intervention. 16S rRNA gene sequencing showed that inulin upregulated the abundance of Akkermansia and Bifidobacterium and downregulated the abundance of Blautia and the Firmicutes:Bacteroidetes ratios, which suggested that inulin could prevent NAFLD via prebiotic effects that suppressed the LPS-TLR4-Mψ pathway in the gut–liver axis.⁴²

Bupleurum Radix (ChaiHu in Chinese): B Radix is a CHM that is used in preventing and treating obesity-related diseases. In an early study in 2012, a water-soluble PS fraction (WBCP) of B Radix roots was purified by DEAE-cellulose and Surperdex 200 HR chromatography. WBCP significantly inhibited the activities of AST, ALT, ALP, and LDH and activated glutathione (GSH), glutathione reductase (GR), γ-glutamylcysteine synthetase (GCS), glutathione S-transferase, and superoxide dismutase, which demonstrated that the hepatoprotective effects of WBCP might be mediated via antioxidant activities.⁷³ In a recent study, Wu et al found that B Radix Extract (BupE) improved hepatic steatosis in HFD mice by activating the FGF21 pathway. Microbiota sequencing results indicated that Bacteroides acidifaciens and Ruminococcus gnavus were involved in the anti-obesity effects of BupE in HFD mice, which suggested that the PS of B Radix might counteract hepatic lipid metabolism disorders by reversing obesity-induced alterations of the microbiota.⁷⁴

Lycium barbarum (GouQiZi in Chinese): L barbarum has been an essential part of CHM as well as dietary use for thousands of years. Studies have reported the health benefits of L barbarum polysaccharide (LBP) on NAFLD.^75,76 Cui et al found that the relative abundance of Lactobacillus and Bifidobacterium increased, but Firmicutes, Actinobacteria, Alistipes, and Clostridiales decreased by LBP.⁷⁷ Zhang et al isolated and purified an acidic heteropolysaccharide, LFP-a1, from L barbarum by hot water extraction, alcohol precipitation, deproteinization, and DEAE-52 cellulose chromatography. They demonstrated that LFP-a1 improved cell viability by inhibiting the oxidative stress in a NAFLD model of L02 cells. In a NAFLD model of zebrafish larvae, LFP-a1 preserved liver integrity by inhibiting abnormal oxidative stress and apoptosis, which suggested that the prebiotic and hepatoprotective effects of LBP might be applied to prevent and treat NAFLD.⁷⁸

Alismataceae : A orientale (ZeXie in Chinese) is a traditional Alismataceae herb that is often used to treat hyperlipidemia. Xu et al found that A orientale significantly decreased serum TC and LDL-C levels in rats. In addition, A orientale particularly promoted the relative abundance of gut microbiota involved in lipid metabolism, such as Lachnospiraceae, which illustrated that the hypolipidemic function of A orientale was mediated mainly through the prebiotic effects.⁷⁹ Deng et al observed the protective effects of SSP, the PS of another Alismataceae herbal, Sagittaria sagittifolia (Cigu in Chinese) against NAFLD in mice. In addition, SSP significantly altered the expression patterns of upstream regulators in the liver, such as Nrf2 and HO-1.⁸⁰

Schisandra chinensis (WuWeiZi in Chinese): S chinensis is a traditional Chinese herbal remedy for liver injury. Aqueous and ethanolic extracts of S chinensis have a significant lipid-lowering effect in NAFLD mice.^81,82 Su et al found that S chinensis polysaccharide significantly improved dysbiosis by upregulating SCFAs.⁸³ In addition, Cheng et al found that S chinensis bee pollen extract (SCPE) reduced fasting blood glucose levels, lipid deposition in the liver, hepatic oxidative damage, and inflammation in HFD mice. SCPE effectively inhibited NAFLD development by suppressing the gene expression of LXR-α, SREBP-1c, and FAS and modulated dysbiosis.⁸⁴

Atractylodes macrocephala (FuLing in Chinese), Poria cocos (BaiZhu in Chinese), and related formulations: In an early study, Meng et al investigated the therapeutic effects of a recipe that contained A macrocephala polysaccharide (ACG) on experimental NAFLD. The results showed that ACG significantly decreased hepatic TG levels in NAFLD rats and mice, and TG production in an in vitro NAFLD model of HepG2 cells that were pretreated with free fatty acid. In addition, ACG improved the hepatic IR and mitochondrial damages in NAFLD mice.⁸⁵ Another recent study conducted by Feng et al revealed that PS from A macrocephala (AMP) extensively modulated dysbiosis and the host's metabolism by upregulating SCFAs production.⁸⁶ Sun et al isolated a water-insoluble polysaccharide (WIP) from P cocos, which is another common CHM.⁴³ NMR and SEC-RI-MALLS analysis revealed that was a (1-3)-β-D-glucan with an average molecular weight of 4.486 × 10⁶ Da. WIP significantly alleviated hepatic hyperglycemia, hyperlipidemia, and steatosis in ob/ob mice by elevating the butyrate levels and activating the PPAR-γ pathway in the gut. 16S rDNA sequencing indicated that WIP increased the abundance of butyrate-producing bacteria Lachnospiracea and Clostridium, which was confirmed by FMT from treated mice.

In addition, several formulations that contain A macrocephala and P cocos have a beneficial effect on NAFLD via their prebiotic properties. For example, Shenling Baizhu powder (SLBZP) is a formulation that contained A macrocephala and P cocos that improves NAFLD clinically. Zhang et al found that SLBZP reduced the serum levels of TC and inflammatory cytokines, and the hepatic expression of the TLR4 pathway proteins in HFD rats. In addition, SLBZP alleviated hepatic steatosis and repaired intestinal integrity. A metagenomics assay revealed that SLBZP increased the relative abundance of SCFAs producing species, which included Bifidobacterium and Anaerostipes.⁸⁷ Ling-gui-zhu-gan decoction (LGZG), another formula that contains A macrocephala and P cocos, has been used to improve steatosis in NAFLD. Liu et al found that FMT from LGZG-treated mice reduced HFD-induced hepatic steatosis and hyperglycemia by modulating gut microbiota.⁸⁸

Maydis stigma (Yumixu in Chinese): M stigma (corn silk) is a well-known medicinal herb that is used in China, Turkey, the United States, and France to treat edema, and cystitis, gout, kidney stones, nephritis, and prostatitis. Recently, the hypolipidemic and hypoglycemic of M stigma polysaccharides (MSP) has been demonstrated in rats by significantly reducing blood glucose, TC, TG, and LDL.⁸⁹ In addition, a corn silk aqueous extract significantly reduced hepatic steatosis, the level of serum TC, LDL, and the hepato-somatic index in HFD rats. The signaling transduction experiments revealed these NAFLD attenuating effects might be partly related to the inhibition of the PI3K/Akt/mTOR pathway in the liver.⁹⁰ Another study discovered that by increasing the dose of MSP, intestinal Lactobacillus and Bacteroides increased in quality and quantity in type-2 diabetic mice, which was consistent with prebiotic characteristics.⁹¹ In combination, these studies indicated that MSP could be considered as a potential candidate to treat NAFLD by restoring the intestinal microflora balance.

The previous studies require some improvements. For example, they focused on the isolated components of Chinese herbal PS and did not reveal the prebiotic effect of their chemical structures. In addition, these studies did not make comparisons between the prebiotic effect of Chinese herbal PS and its direct effect on NAFLD. Moreover, the indicators used to assess the pathogenesis of NAFLD in the animal models varied. Several studies examined liver pathological damage, such as hepatic lipid deposition and hepatic steatosis, and others examined liver weight gain and TG, TC, AST, and ALT levels.

Discussion and Perspective

In summary, these studies indicated that PS derived from CHMs might have several beneficial effects when used as prebiotics in the treatment of NAFLD. First, a single PS of CHM might contain a variety of prebiotic ingredients, and therefore, constitute a multivalent prebiotic formulation. For example, as mentioned previously, intaking LBP orally increases the abundance of Lactobacillus and Bifidobacterium, when decreasing Firmicutes, Actinobacteria, Alistipes, and Clostridiales in mice guts.⁷⁷ In addition, it has been consistently demonstrated that after 24 h fermentation by the gut microbiota, the degraded LBP can increase the relative abundance of Bacteroides, Bifidobacterium, Phascolarctobacterium, Clostridium XlVb, Prevotella, and Collinsella, which implies that the gut microbiota degradation products of LBP could act as a prebiotics combination to reverse dysbiosis.²⁷ Second, to effectively treat a variety of significant chronic inflammatory diseases, such as NAFLD, prebiotics should achieve homeostasis of microbiota and improve abnormal cell metabolism or damage. Therefore, the development of next-generation multifunctional prebiotics for specific diseases is critical.^92,93 As given in Table 1, the studies described previously found that Chinese herbal PS directly regulated the gut microbiota and exerted hepatoprotective effects, such as anti-oxidation, hypolipidemia, and anti-inflammation via the gut–liver axis, which meets the requirements of the new generation of prebiotics and reaffirms the holistic benefit of CHMs. In addition, Chinese herbal PS should be safer than probiotics, which are live microorganisms that provide health benefits. The overall risk of infection is low and comparable between probiotics and commensal bacterial strains. However, certain immunocompromised, malnourished, and cancer patients must exercise additional caution when using probiotics to avoid infections that are caused by probiotics and other potentially pathogenic bacteria.^94,95 By directly detecting the distribution of intestinal flora, Zmora et al found that standard probiotic bacteria could not successfully colonize the digestive tracts of two-thirds of subjects, termed “resisters” by the authors. They discovered that resisters expelled the ingested microbiota, and only a small number of subjects, known as “persisters,” were successful in colonizing the gastrointestinal tract with probiotics. In addition, the individualized gut mucosal colonization capacity was correlated with baseline host transcriptional and microbiome characteristics; however, not with the stool levels of probiotics during consumption.⁹⁶ In comparison, CHM has been practiced for thousands of years, and many of the herbs, such as G lucidum, P multiflorum, B Radix, L barbarum, and A orientale, could be used as medicinal foods with confidence.

According to the multiple parallel hits hypothesis, NAFLD pathogenesis and progression are triggered by a central pathway of aberrant innate immunity, which is accompanied by stress signaling networks and circulating proinflammatory cytokines.⁹⁷ This theory has resulted in the clinical translation of a number of potential therapeutics that target various aspects of metabolic disruption, oxidative stress or inflammatory signaling, or both.⁹⁸ However, with no FDA-approved drugs, current NAFLD management has to primarily rely on dietary control, lifestyle modifications, physical activity, bariatric surgery, and various existing pharmacological approaches, which include CHMs, particularly in China. For Chinese herbal PS that have demonstrated efficacy and safety in experimental NAFLD, the focus should be on modifying their structure or developing new dosage forms to enhance their pharmacokinetic properties, and therefore, expand the range of clinical medications available. In addition, to advance research into the prevention and treatment of NAFLD using Chinese herbal PS, researchers should attempt to combine the in vitro cell model and the in vivo animal model to complement each other.⁹⁹ Furthermore, clinical trials should be conducted to determine the efficacy and safety of PS from CHMs in the treatment of NAFLD. Our laboratory has laid the groundwork for several Chinese herbal PS. In the future, additional research will be conducted to determine the prebiotic effect of these PS on further advances in NAFLD treatment.

Footnotes

Author Contributions

L.J. and Y.K. wrote and edited the manuscript. Z.Y., B.J., and S.Y. participated in the writing process. L.L. and J.L. revised and finalized the manuscript. All authors contributed to the article and approved the submitted version.

Author's Note

Yu-Na Kan is also affiliated with Department of Food Safety and Nutrition, School of Basic Medical Sciences, Guizhou University of Traditional Chinese Medicine, Guiyang, China. Zhi-Pu Yu is also affiliated with Department of Equipment, The Second Affiliated Hospital, Qiqihar Medical University, Qiqihar, China. Bai-Yu Jian is also affiliated with Department of Polygenic Diseases, Research Institute of Medicine and Pharmacy, Qiqihar Medical University, Qiqihar, China. Ji-Cheng Liu is also affiliated with Department of Natural Drug Antitumor, Research Institute of Medicine and Pharmacy, Qiqihar Medical University, Qiqihar, China.

Data Availability

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics and Patient Consent

We confirm that Ethical Committee approval is not relevant to our manuscript, since this is a Review.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Science Project of Qiqihar Medical University (grant number QMSI2019Z-02).

ORCID iD

Li-Yan Jiang

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