HHQ16 Alleviates Nonalcoholic Fatty Liver Disease Via Supressing Liver Ferroptosis

1.Background and purposeNonalcoholic fatty liver disease（NAFLD）is defined as the fatty liver with metabolic dysfunction.Nearly 1 billion people are affected withNAFLD globally,causing a huge economic burden to society.However,there has no approved drug therapy for NAFLD.Traditional Chinese medicine（TCM）have been widely used in hepatic diseases for centuries in Asia,and clinical trials with TCM therapy for NAFLD have shown a good prospect.Astragali radix,also named Huang qi,is the A.membranceus（Fisch.）Bge.var.mongholicus（Bge.）Hsiao（radix astragali）.Astragaloside IV is one of the main bioactive constituents of astragali radix,which is reported to reduce deposition as well as inflammation in liver.However,it has a poor druggability.HHQ16 is modified from astragaloside IV,which has a better solubility in water,indicating a better druggability and help to develop a new drug.The aim of this research is to study the pharmacodynamics and mechanism of HHQ16 on NAFLD.2.MethodIn vivo,three commonly models for NAFLD,including 60%high fat diet（HFD）-fed mice,leptin deficiency mice（ob/ob）and methionine and choline deficent diet（MCD）-fed mice,were used to evaluate pharmacodynamic of HHQ16 on NAFLD.0.5%CMCNa was used as negative control,while obeticholic acid was positive control.All drugs were administered by intragastric administration for 4 weeks after modeled.Then liver weight,liver function and histopathological evaluation were assessed.In vitro,free fatty acid（FFA）solution was used treat AML12 cells,which can simulate lipid deposition and injury in vivo.Then,the level of triglycerides,cell viability and oil red o staining were applied to evaluate effect of HHQ16.To study the mechanism of HHQ16 on NAFLD,RT-q PCR,Western blot and ELISA were used to detect the expression of indicated targets and indexes related to ferroptosis.Furthermore,ORM1 knockout mice was used to clarify the role of ORM1 in ferroptosis and NAFLD, combined with transcriptomics,bioinformatics and protein detection.At last,the expression of ORM1 was knocked out or knocked down to valiade whether it mediated the effect of HHQ16 on ferroptosis and NAFLD.3.Results（1）HHQ16 can improve NAFLDIn all three models,HHQ16 reduced NAS score,lipid deposition,inflammation and the liver function in a dose dependent manner.Furthermore,the effect of HHQ16 on NAFLD is superior to OCA in MCD mice.In vitro,HHQ16 improved intracellular lipid deposition,lipid damage and increased cell viability of AML12 cells treated with FFA.（2）HHQ16 can suppress ferroptosis in NAFLDTo explore how HHQ16 exerts hepatoprotective effect,the representative proteins of various PCDs were detected in all three rodent models.Results showed that HHQ16 upregulated ferroptosis-related GPX4 in all models.In addition,HHQ16 influenced various indexs related to ferroptosis,including ferrous iron(Fe²⁺),glutathione（GSH）,long chain fatty acid Co A ligase 4（ACSL4）and GPX4,malondialdehyde（MDA）,4-hydroxynomenal（4-HNE）and lactic dehydrogenase（LDH）.（3）HHQ16 can suppress ferroptosis via ORMPrevious studies have found that ORM plays an important role in energy metabolism,metabolic-related diseases,which also is a potential target for drug in NAFLD.This study found that,HHQ16 can not upregulate ORM expression in L02 and AML12 cells with a time and dose-dependent manner,but also in three rodent models.HFD-fed,ORM1-/-mice display more serious NAFLD compared to ORM1+/+mice.The analysis of liver transcriptomics between ORM1+/+and ORM1-/-mice showed that ORM1 deficiency is closely relative to ferroptosis.After ORM1 was silenced by si RNA in hepatocytes,HHQ16cannot increase the expression of GPX4,indicating that ORM mediated the suppression of HHQ16 on ferroptosis.（4）HHQ16 can suppress ferroptosis and improve NAFLD via ORMAfter fed by MCD for 4 weeks,HHQ16 was administered by intragastric administration to ORM1+/+and ORM1-/-mice for another four weeks.The results showed that the effect of HHQ16 on ferroptosis suppression,hepatoprotection and NAFLD improvement were abolished in ORM1-/-mice,indicating that ORM1 is essencial for HHQ16 to exert its effects on ferroptosis and NAFLD.4.ConclusionIn conclusion,HHQ16 upregulated ORM expression in liver,thereby suppress ferroptosis and ultimately improve NAFLD.