ObjectiveThe study aims to investigate the intervention effect of modified Xiangsha Liujunzitang （M-XSLJZ） on intestinal-derived lipopolysaccharide （LPS）-activated Kupffer cell inflammation in rats with hyperlipidemia spleen deficiency syndrome. MethodsSeventy male SD rats were randomly divided into seven groups （n=10）： blank control （CON）， high-fat diet without spleen deficiency （HFD）， high-fat diet with spleen deficiency （SD-HFD）， M-XSLJZ low-， medium-， and high-dose groups （XS-L， XS-M， XS-H）， and western medicine control （R）. Spleen deficiency was induced in SD-HFD， XS-L， XS-M， XS-H， and R groups via irregular diet combined with exhaustive swimming for 15 days. The CON group received a standard diet， while other groups were fed a high-fat diet for 10 weeks to establish the hyperlipidemia model. After successful modeling， rats were treated for 8 weeks： M-XSLJZ was administered at 3.51， 7.02， 14.04 g·kg^-1 in XS-L， XS-M， and XS-H groups， respectively. The R group received 9×10^-4 g·kg^-1 of a reference drug. D-xylose excretion rate was measured by the phloroglucinol method. Blood lipids were assessed using an automated biochemical analyzer. Hematoxylin-eosin （HE） staining was used to evaluate the pathological conditions of the liver， and oil red O staining was used to observe the lipid deposition in the liver. The levels of LPS， portal vein serum LPS， LPS-binding protein （LBP）， serum interleukin-6 （IL-6）， IL-1β， and tumor necrosis factor-α （TNF-α） were detected by enzyme-linked immunosorbent assay （ELISA）. Immunofluorescence was used to evaluate CD86 expression and CD68/TLR4 co-localization in the liver. Protein levels of TLR4， MyD88， NF-κB p65， and p-NF-κB p65 in Kupffer cells were analyzed via Western blot automated protein analysis. Hepatic IL-6， TNF-α， and IL-1β mRNA and protein levels were measured using Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot. ResultsCompared with the CON group， the SD-HFD group showed a decrease in D-xylose excretion （P<0.01）. TC， TG， HDL-C， and LDL-C increased （P<0.05， P<0.01）. A large number of hepatic lipid vacuoles and orange-red lipid droplet deposition appeared in the liver. Ileal LPS， portal LPS， and LBP increased （P<0.05， P<0.01）. The levels of serum IL-6， TNF-α， and IL-1β increased （P<0.01）. The expression of CD86 was upregulated （P<0.01）， and the co-expression of CD68 and TLR4 was enhanced. The protein levels of TLR4， MyD88， and p-p65 in Kupffer cells increased （P<0.01）. The mRNA and protein levels of IL-6， TNF-α， and IL-1β increased （P<0.05， P<0.01）. Compared with the HFD group， the SD-HFD group exhibited decreased D-xylose excretion （P<0.01）， higher HDL-C， LDL-C （P<0.05）， increased portal LBP and LPS （P<0.05）， increased serum IL-6 and TNF-α （P<0.01）， upregulated CD86 （P<0.01）， enhanced CD68/TLR4 co-expression， and higher TNF-α mRNA/protein （P<0.05）. Compared with the SD-HFD group， all M-XSLJZ treatment groups showed reduced TC， TG， and LDL-C （P<0.05， P<0.01）. XS-H and R groups displayed improved hepatic lipid deposition. XS-H and R groups had lower ileal LPS， portal LPS， and LBP levels （P<0.05， P<0.01）. All M-XSLJZ treatment groups exhibited reduced serum IL-6， IL-1β， and TNF-α （P<0.01）. The XS-H group showed downregulated CD86 （P<0.01） and weakened CD68/TLR4 co-expression. The XS-H group had reduced TLR4， MyD88， and p-NF-κB p65 in Kupffer cells （P<0.01）. XS-H and R groups showed lower IL-6， TNF-α， and IL-1β mRNA/protein （P<0.05， P<0.01）. ConclusionM-XSLJZ may exert its lipid-lowering effects by inhibiting intestinal-derived LPS and alleviating Kupffer cell inflammation in the liver.

OBJECTIVE: To explore the improvement effect of electroacupuncture (EA) based on Xingnao Kaiqiao acupuncture (acupuncture for regaining consciousness and opening orifices) on cognitive impairment in mice with Parkinson's disease (PD), and to explore its regulatory mechanisms on the kinesin family member 5A (KIF5A)/mitochondrial Rho GTPase 1 (Miro1) pathway and mitophagy in prefrontal cortical neurons. METHODS: A total of 70 male C57BL/6J mice of clean grade were randomly divided into a normal group (12 mice), a sham operation group (12 mice), and a model pre-screening group (46 mice). Unilateral stereotaxic injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle was adopted to establish the PD model in the model pre-screening group. Twenty-four mice after successful modeling were randomly selected and divided into a model group and an EA group, 12 mice in each one. In the EA group, acupuncture was applied at "Shuigou" (GV26) and bilateral "Sanyinjiao" (SP6) and "Neiguan" (PC6), ipsilateral "Sanyinjiao" (SP6) and "Neiguan" (PC6) were connected to EA respectively, with disperse-dense wave, 5 Hz/20 Hz in frequency, 0.5 mA in current intensity, 20 min a time, 6 times a week for 30 days. Cognitive function was assessed by Y-maze and Morris water maze tests; morphology of prefrontal cortex was observed by H.E. staining; reactive oxygen species (ROS) level in prefrontal cortex was detected by fluorescence probe method; mitochondrial morphology and autophagosome ultrastructure were observed by transmission electron microscopy; the mRNA expression of tyrosine hydroxylase (TH) was detected by quantitative real-time PCR; the protein expression of TH, KIF5A, Miro1, p62, Parkin and PTEN induced kinase 1 (PINK1) was detected by Western blot. RESULTS: Compared with the sham operation group, both the model group and the EA group exhibited increased rotation number of per minute (P<0.001). Compared with the sham operation group, in the model group, the novel arm exploration time of Y-maze test was shortened (P<0.001), the escape latency of Morris water maze test was prolonged (P<0.05) and the platform crossing number of Morris water maze test was reduced (P<0.01); in the prefrontal cortex, the number of cellular vacuole and neurons with karyopyknosis was increased (P<0.001), and mitochondrial autophagosomes could be observed; in the prefrontal cortex, the relative expression of ROS was increased (P<0.001), the protein and mRNA expression of TH was decreased (P<0.001), the protein expression of Miro1, PINK1, Parkin was increased (P<0.001, P<0.01), the protein expression of KIF5A and p62 was decreased (P<0.001). Compared with the model group, in the EA group, the novel arm exploration time of Y-maze test was prolonged (P<0.01), the escape latency of Morris water maze test was shortened (P<0.05) and the platform crossing number of Morris water maze test was increased (P<0.05); in the prefrontal cortex, the number of cellular vacuole and neurons with karyopyknosis was decreased (P<0.001), and the number of mitochondrial autophagosomes reduced and the mitochondrial morphology was improved; in the prefrontal cortex, the relative expression of ROS was decreased (P<0.01), the protein and mRNA expression of TH was increased (P<0.001, P<0.01), the protein expression of Miro1, PINK1, Parkin was decreased (P<0.001, P<0.01, P<0.05), the protein expression of KIF5A and p62 was increased (P<0.01, P<0.05). CONCLUSION Xingnao Kaiqiao electroacupuncture effectively alleviates cognitive impairment and damage of neuronal function in PD mice, its mechanism may be related to the regulation of KIF5A/Miro1 pathway, hence reducing the mitophagy in prefrontal cortical neurons.
Animals ; Electroacupuncture ; Male ; Mice ; Parkinson Disease/physiopathology* ; Cognitive Dysfunction/psychology* ; Kinesins/genetics* ; Humans ; Mitophagy ; Mice, Inbred C57BL ; rho GTP-Binding Proteins/genetics* ; Mitochondria/genetics* ; Prefrontal Cortex/metabolism*

OBJECTIVE: To investigate the effect of stretch on long non-coding RNA taurine upregulated gene 1 (TUG1)-mediated miR-545-3p/cannbinoida receptor 2 (CNR2) pathway regulating bone regeneration in the distraction area of rats during distraction osteogenesis. METHODS: Thirty-six 10-week-old male Sprague Dawley rats were randomly divided into 3 groups ( n=12 in each group): group A (femoral fracture+injection of interfering RNA), group B (distraction osteogenesis+injection of interfering RNA), and group C (distraction osteogenesis+injection of TUG1). Groups A and B were injected with 60 μg of interfering RNA at the beginning of incubation period (immediate after operation), the beginning of distraction phase (7 days after operation), and the end of distraction phase (21 days after operation), and group C was injected with 60 μg of synthetic TUG1 in vivo interfering sequence at the same time. The general situation of rats in each group was observed during the experiment. The mineralization of fracture space or distraction area was observed by X-ray films at 21, 35, and 49 days after operation. At 49 days after operation, the samples of the distraction area were taken for HE staining to observe the mineralization, and real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expressions of osteoblast-related genes such as TUG1, miR-545-3p, CNR2, alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). Blood samples were collected from the abdominal aorta of the rats, and the expressions of ALP and C terminal telopeptide of type Ⅰ (CTX-Ⅰ) protein were detected by ELISA assay. RESULTS: The results of X-ray film and HE staining observations showed that osteogenesis in group C was superior to groups A and B at the same time point. The results of qRT-PCR showed that the relative mRNA expressions of TUG1, CNR2, ALP, OCN, and OPN in group C were significantly higher than those in group A and group B, and the relative mRNA expression of miR-545-3p in group C was significantly lower than that in group A and group B ( P<0.05). The relative mRNA expressions of TUG1 and ALP in group B were significantly higher than those in group A, and the relative mRNA expression of miR-545-3p in group B was significantly lower than that in group A ( P<0.05). There was no significant difference in the relative mRNA expressions of CNR2, OCN, and OPN between group A and group B ( P>0.05). The results of ELISA showed that the expressions of ALP and CTX-Ⅰ protein were significantly higher in group C than in group A and group B, and in group B than in group A ( P<0.05). CONCLUSION Under the action of stretch, the expression of TUG1 in the femoral distraction area of rats increases, which promotes the expression of CNR2 by inhibiting the expression of miR-545-3P, which is helpful to the mineralization of the extension area and osteogenesis.
Animals ; MicroRNAs/genetics* ; Rats, Sprague-Dawley ; Male ; Osteogenesis, Distraction/methods* ; Rats ; RNA, Long Noncoding/metabolism* ; Osteopontin/genetics* ; Osteogenesis ; Bone Regeneration ; RNA, Small Interfering/genetics* ; Osteocalcin/genetics* ; Alkaline Phosphatase/metabolism* ; Osteoblasts/cytology* ; Signal Transduction ; Femoral Fractures/surgery*

Viruses constitute a significant group of pathogens that have caused numerous fatalities and substantial economic losses in recent years, particularly with the emergence of coronaviruses. While the impact of SARS-CoV-2 appears to be diminishing in daily life, only a limited number of drugs have received approval or emergency use authorization for its treatment. Given the high mutation rate of viral genomes, host-directed agents (HDAs) have emerged as a preferred choice due to their broad applicability and lasting effectiveness. In contrast to direct-acting antivirals (DAAs), HDAs offer several advantages, including broad-spectrum antiviral activities, potential efficacy against future emerging viruses, and a lower likelihood of inducing drug resistance. In our review article, we have synthesized known host-directed antiviral targets that span diverse cellular pathways and mechanisms, shedding light on the intricate interplay between host cells and viruses. Additionally, we have provided a brief overview of the development of HDAs based on these targets. We aim for this comprehensive analysis to offer valuable perspectives and insights that can guide future antiviral research and drug development efforts.

Objective To explore the expression profile of adenosine A1 receptor in the paraventricular thalamic nucleus(PVT)in wakefulness/sleep state and its role in regulating sleep.Methods Male C57BL/6J mice(6～12 weeks old,weighing 22～28 g)were randomly divided into ZT0,ZT6,ZT10,ZT12,and sleep deprivation and recover0y sleep groups(n=16 or n=4).RT-qPCR and fluorescence in situ hybridization were used to observe the changes of adenosine A1 receptor in PVT.Whole-cell patch-clamp recording was conducted to determine the effects of adenosine on the membrane potential and discharge frequency of PVT neurons.The experimental animals were divided into adenosine A1 receptor interference group(n=8)and interference control group(n=7).After RNA interference,electroencephalography(EEG)and electromyography(EMG)were applied simultaneously to observe the changes in wake and sleep time between the 2 groups during sleep deprivation for 6 h and sleep recovery for 4 h.Results RT-qPCR and fluorescence in situ hybridization confirmed that the expression of adenosine A1 receptor in PVT was affected by wakefulness/sleep state,with the level in the ZT0 group(active stage)significantly higher than that in the ZT12 group(non-active stage,P<0.05).Whole-cell patch clamp recording indicated that adenosine exerted inhibitory effects on PVT neurons through 2 distinct response types(P<0.05),and the inhibition was in a diminishing trend with a decrease in the expression level of adenosine A1 receptor.After sleep deprivation,the expression level of adenosine A1 receptor showed significant intergroup differences:the level was significantly higher in the sleep deprivation group than the recovery sleep group(P<0.05),while that of the recovery sleep group was higher than that of the ZT6 group and that of the ZT10 group(P<0.05).After knocking down adenosine A1 receptor(shRNA-A1R)in the PVT,the wakefulness timing of the shRNA-A1R group was significantly increased,while the sleep timing was significantly decreased within 1 h after sleep recovery when compared with the interference control group(P<0.05).Conclusion The expression of adenosine A1 receptor in the PVT is dynamically regulated by sleep pressure,which was increasing as sleep pressure rises.

Objective To investigate the potential mechanism by which dihydromyricetin(DHM)ameliorates diabetic nephropathy(DN),and to screen and validate its possible key molecular targets.Methods A DN model was established using db/db mice,and 100 mg/(kg·d)DHM was administered via gavage 5 d per week for totally 10 weeks.Renal morphological changes were observed after staining to evaluate the effects of DHM.GSE161885 and GSE270526 datasets were obtained from the Gene Expression Omnibus(GEO)database and analyzed in combination with the GeneCards database to screen for DN-related differentially expressed genes(DEGs).Protein-protein interaction(PPI)network and molecular docking were employed to predict potential DHM targets.Western blotting and immunofluorescence staining were performed to detect the effects of DHM on pyroptosis-related pathways in the renal tissues of db/db mice and in high glucose(HG)-induced human renal tubular epithelial cells(HK-2).The specific NLR family pyrin domain containing protein 3(NLRP3)inhibitor MCC950 was also used to validate the predicted mechanism.Results In vivo experiments showed that DHM significantly ameliorated renal pathological damage in db/db mice,alleviated glomerular hypertrophy and mesangial expansion,and markedly reduced Paller scores(P<0.001).Immunofluorescence staining revealed significantly weakened fluorescence signals for α-smooth muscle actin(α-SMA),fibronectin,and collagen Ⅰ in renal tissues.Western blot results showed that the expression levels of collagen Ⅰ,collagen Ⅲ,α-SMA,and transforming growth factor beta 1(TGF-β1)were significantly decreased(P<0.05).A total of 16 DN-related DEGs were identified.Enrichment analysis revealed that these genes were primarily enriched in pathways such as viral protein interactions,cytokine-cytokine receptor interaction,and the AGE-RAGE signaling pathway in diabetic complications,and were primarily involved in gene functions such as the positive regulation of lymphocyte-mediated immunity,positive regulation of adaptive immune response,and chemokine activity.Molecular docking confirmed NLRP3 as a potential target of DHM.In vivo validation showed that DHM significantly down-regulated gasdermin-D(GSDMD)fluorescence signals and inhibited the expression of pyroptosis-related proteins including NLRP3,Caspase 1,Cleaved-Caspase 1,interleukin 18(IL-18),and GSDMD(P<0.05).In vitro studies further confirmed that both DHM and the specific NLRP3 inhibitor MCC950 alleviate high glucose-induced fibrosis and pyroptosis in HIC-2 cells.Conclusion DHM can ameliorate the progression of DN,and its mechanism is related to inhibiting NLRP3 inflammasome-mediated pyroptosis,thereby alleviating renal inflammation and fibrosis.

Background Long-term exposure to noise during sleep may has adverse effects on metabolic system, and liver lipid metabolism is closely related to circadian clock genes. Objective To investigate the effects of long-term noise exposure during sleep on liver circadian clock and lipid metabolism in mice and its related mechanism. Methods Twenty C57BL/6J male mice were randomly divided into two groups: a noise exposure group and a control group with 10 mice in each group. The mice in the noise exposure group were exposed to white noise at 90 dB sound pressure level (SPL) for 30 consecutive days, 8 h a day, from 9:00 to 17:00. The mice in the control group were exposed to background noise ≤40 dB SPL. After noise exposure, the animals were neutralized at 14:00 (ZT6) and 2:00 (ZT18), 5 animals at each time spot, and the liver tissues were collected. Total cholesterol and triglyceride in liver were determined by cholesterol oxidase method and glycerol phosphate oxidase method respectively. The expressions of circadian clock genes (Clock, Bmal1, Rev-erbα, and Rev-erbβ) and lipid metabolism genes (Srebp1c, Hmgcr, Fasn, Lxrα, Acc1, and Chrebp) in liver were detected by quantitative real-time PCR. Results Compared with the control group, the content of total cholesterol in liver in the noise exposure group increased by 48% (P<0.05) and the content of liver triglyceride increased by 61% (P<0.05) at ZT18. The mRNA expression levels of circadian clock genes Clock and Bmal1 in the noise exposure group was significantly increased at ZT18 and decreased at ZT6 (P<0.05). The mRNA expression level of Rev-erbα decreased at both ZT6 and ZT18 (P<0.05). The mRNA expression level of Rev-erbβ had no significant change at ZT6 and ZT18. The mRNA expression levels of liver lipid metabolism related genes Srebp1c, Hmgcr, Chrebp, and Lxrα in the noise exposure group were higher than those in the control group at ZT18 (P<0.05). The mRNA expression levels of Acc1 and Fasn showed no significant change at ZT6, then an upward trend at ZT18, but no significant difference between the two time spots (P>0.05). Conclusion Long-term noise exposure during sleep can cause circadian clock and lipid metabolism disorders in mice. Among them, suppression of key circadian clock genes may be associated with Rev-erbα-mediated upregulation of the nuclear receptors Srebp1c and Chrebp for lipid synthesis and deposition in the liver, resulting in lipid metabolism disorder.

ObjectiveTo investigate the mechanism of Xuefu Zhuyu capsules against atherosclerosis via regulating polarization of macrophages based on Notch1/jagged canonical Notch ligand 1（Jagged1）/Hes family BHLH transcription factor 1（Hes1） signaling pathway. MethodThe mouse models with atherosclerosis were prepared by feeding the mice with an ApoE^-/- high-fat diet for four weeks， and they were randomly divided into the model group， Xuefu Zhuyu capsule group， and atorvastatin group. C57BL/6 mice were fed as a normal group. The Xuefu Zhuyu capsule group was intragastrically given Xuefu Zhuyu capsules （0.728 g·kg^-1·d^-1）， and the atorvastatin group was intragastrically given atorvastatin tablet （6.07 mg·kg^-1·d^-1）. The normal group and the model group were given equal volume of the deionized water by intragastric administration， and the intervention lasted for 12 weeks. Aortic plaque morphology was observed by hematoxylin-eosin （HE） staining， and aortic plaque area and lipid deposition were observed by oil red O staining. The positive expression levels of CD86 and CD206 in aortic tissue were detected by immunohistochemistry， and serum levels of tumor necrosis factor （TNF）-α， interleukin（IL）-1β， transforming growth factor （TGF）-β₁， and IL-10 were detected by enzyme-linked immunosorbent assay （ELISA）. The relative mRNA expressions of inducible nitric oxide synthase （iNOS）， arginase-1 （Arg-1）， Notch1， Jagged1， and Hes1 in aortic tissue were detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The relative protein expression of iNOS， Arg-1， Notch1， Jagged1， and Hes1 in aortic tissue was detected by Western blot. ResultCompared with the normal group， the model group had significant aortic plaque and lipid deposition， and the expression levels of pro-inflammatory cytokines TNF-α and IL-1β were increased （P<0.01）. The expression level of anti-inflammatory cytokine TGF-β₁ showed a downward trend， but the difference was not statistically significant. The mRNA and protein expressions of iNOS were increased （P<0.01）. The protein expression of Arg-1 was decreased （P<0.01）， and the mRNA expression of related pathway molecule Jagged1， as well as the protein expressions of Notch1， Jagged1， and Hes1 were increased in the model group （P<0.05， P<0.01）. Compared with those in the model group， the plaque area and lipid deposition had a decreasing trend in the Xuefu Zhuyu capsule group， and the expressions of TNF-α and IL-1β showed a downward trend. The expression of TGF-β₁ was increased （P<0.05）， and the expression of macrophage marker CD86 was decreased. The mRNA and protein expressions of iNOS were decreased （P<0.01）. The mRNA and protein expressions of Arg-1 were increased （P<0.05， P<0.01）. Furthermore， the mRNA and protein expressions of Notch1， Jagged1， and Hes1 were decreased （P<0.01）. ConclusionXuefu Zhuyu capsules can reduce aortic plaque area and lipid deposition in mice with atherosclerosis， alleviate inflammation， inhibit M1 macrophages， and promote the expression of M2 macrophages， and the mechanism may be related to the regulation of Notch1/Jagged1/Hes1 signaling pathway.

OBJECTIVE To construct an insulin-resistant(IR)small intestinal organoid model of mice and study the protective effect of flavanomarein(FM)on the intestinal mucosal barrier in the model.METHODS ①Small intestinal organoid models of C57BL/6J and db/db of mice were constructed.The expressions of Ki-67,E-cadherin(E-cad),lysozyme(Lyz)and mucin-2(Muc-2)in small intestinal organ-oids were detected by 3D immunofluorescence.RT-qPCR was used to detect the expressions of fibro-nectin(Fn),glucagon-like peptide-1(GLP-1)and peotide YY(PYY)mRNA while Western blotting was used to detect the expressions of Fn,GLP-1 and PYY protein.The Lyz secretion level was detected by ELISA.② Small intestinal organoids were divided into five groups:C57BL/6J mice 'small intestinal organ-oids as the normal control group,db/db mice' intestinal organoids as the IR model group,db/db mice small intestinal organoids with flavanomarein 25,50 and 100 μmol·L-1 intervention for 48 h as IR model+ FM groups.RT-qPCR was used to detect the expression of Lyz mRNA while Western blotting was used to detect the expression of Lyz protein.RESULTS ① On the 6th day of small intestinal organoid culture,a ring structure with a clear luminal structure was formed and an IR mouse small intestinal organoid model was established.3D Immunofluorescence detection showed that the established small intestinal organoids all expressed Ki-67,E-cad,Lyz and MUC-2.Compared with the normal control group,the expres-sion of Fn mRNA in the IR model group was significantly increased(P<0.05)while the expressions of GLP-1 and PYY mRNA were significantly decreased(P<0.05).Compared with the normal control group,the expression of Fn protein in the IR model group was significantly decreased(P<0.05)while the expressions of GLP-1 and PYY protein were significantly increased(P<0.05).ELISA results showed that compared with the normal control group,the secretion levels of Lyz in the IR model group were signifi-cantly decreased(P<0.01).② RT-qPCR results showed that compared with the normal control group,the expression of Lyz mRNA in the IR model group was significantly decreased(P<0.01).Compared with the IR model group,the expression of Lyz mRNA in the IR model+FM 50 and 100 μmol·L-1 groups was significantly increased(P<0.05,P<0.01).Western blotting results showed that compared with the normal control group,the expression of Lyz protein in the IR model group was significantly decreased(P<0.01).Compared with the IR model group,the expression of Lyz protein in the IR model+FM 50 and 100 μmol·L-1 groups was significantly increased(P<0.05,P<0.01).CONCLUSION The constructed IR mouse small intestinal organoid model provides a more complete in vitro research model for exploring the pathophysiological mechanism by which drug interventions help repair the intestinal mucosal barrier.FM may maintain the intestinal mucosal barrier by reversing the decrease in Lyz expression levels in IR mice,thereby improving IR.

Apolipoprotein H(APOH)is a glycoprotein synthesized by liver cells,closely related to insulin resistance(IR)and lipoprotein metabolism,and plays an important role in the occurrence and development of diabetes mellitus and dyslipidemia.This article reviews the research progress on the biological functions of APOH and its correlation with metabolic diseases.