Anti-Inflammatory and Therapeutic Effects of a Novel Small-Molecule Inhibitor of Inflammation in a Male C57BL/6J Mouse Model of Obesity-Induced NAFLD/MAFLD

Figures & data

Figure 1 Simplified schematic diagram of the pathologic cycle in obesity-induced NAFLD/MAFLD resulting from multiple parallel hits.

Notes: In a state of obesity, there is an over-abundance of circulating free fatty acids (FFAs) that are released from adipose tissue and absorbed in the gut. There is also a pathologic shift in the gut microbiome (ie, gut microbiome dysbiosis) that leads to an inflammatory state in the gut (in part via activation of TLR4 signaling by gut derived FFAs and bacterial LPS). Gut inflammation then leads to a decrease in gut tight junction proteins (such as OCLN), which causes a disruption in the gut protective barrier (ie, leaky gut). The leaky gut then leads to the release of increased levels of LPS, FFAs, inflammatory proteins (ie, cytokines and chemokines), and other metabolites into the portal vein, where it then goes to the liver. In the liver, these substances cause hepatic inflammation (in part via activation of TLR4 signaling in both hepatocytes and resident immune cells). This hepatic inflammation then leads to an increase in hepatic steatosis (via modulation of lipid metabolism in the liver), hepatocellular ballooning, hepatic insulin resistance, and other liver injury that is associated with NAFLD/MAFLD. In parallel, the increase in chronic, systemic circulating levels of LPS, FFAs, inflammatory proteins, and other metabolites also leads to the activation of inflammation (again, in part, via activation of TLR4 signaling) in adipose tissue (in both adipocytes and associated macrophages/immune cells). This also contributes to the overall state of chronic inflammation systemically in the obese individual. This chronic systemic inflammation is the cause of whole-body insulin resistance that occurs in NAFLD/MAFLD. This is certainly not a complete, comprehensive schematic, but encompasses the main overall processes in the pathogenesis of obesity-induced NAFLD/MAFLD that are relevant to this study.

Figure 2 Derivation and structure of IOI-214 from C10.

Notes: IOI-214 is a small organic compound that was derived from a phenyl derivative of methimazole termed C10. In an attempt to generate compounds that had more potent anti-inflammatory activity than C10, we generated a library of small organic molecules derived from C10 (see scheme above) in which a large number of aryl groups (Ar), substituted alkyl groups (R), and different heteroatoms (X, Y) were inserted into the ring system of C10. The molecular structures of C10 and IOI-214 are shown here and were drawn using the ChemDraw program (Perkin Elmer Informatics, Waltham, MA, USA). The structure of IOI-214 used in these studies was verified by both ¹H-NMR and ¹³C-NMR and was shown to be 98% pure by HPLC.

Figure 3 Synthesis of 3-propyl-4-(pyridin-3-yl)thiazole-2(3H)-thione (IOI-214).

Notes: To a stirred solution of the amine 1 (0.22 mL, 2.67 mmol) in a 1:1 mixture of H₂O/EtOH (9 mL) was added CS₂ (0.32mL, 5.34mmol) and K₂CO₃ (0.495 g, 3.56 mmol). The reaction was stirred for 5 min and 3-(2-bromoacetyl) pyridine hydrobromide (1) (0.503 g, 1.78 mmol) was added and stirred for an additional 3 h. The yellow solid was filtered and dried under vacuum overnight to provide 0.36 g, (80%) of 4-hydroxy-3-propyl-4-(pyridin-3-yl)thiazolidine-2-thione (2), R_f = 0.28 (2% CH₃OH in CH₂Cl₂) mp 129–131 °C; ¹H NMR (CD₃OD, 500 MHz) δ 8.68 (s, 1H) 8.54 (d, J = 4.9, 1H) 7.91 (d, J = 7.8, 1H) 7.51 (m, 1H) 3.67 (s, 1H) 3.11 (s, 1H), 1.60 (m, 2H) 0.79 (t, J = 7.3, 3H). To the crude alcohol (2) (200 mg, 0.78 mmol) in a dry flask was added a freshly prepared solution of anhydrous HCl (1 M in EtOAc, 2.0 mL) and dry EtOH (1.6 mL). The reaction mixture was refluxed for 24 h. TLC confirmed reaction completion and the solvent was removed under vacuum to provide 190 mg (100%) of IOI-214 as a dark yellow solid, R_f = 0.62 (2% CH₃OH in CH₂Cl₂) mp 175–177 °C; ¹H NMR (CD₃OD, 500 MHz) δ 9.16 (s, 1H), 9.05 (d, J = 4.0, 1H), 8.79 (d, J = 7.4, 1H), 8.26 (t, J = 6.9, 1H), 7.20 (s, 1H) 4.15 (t, J = 7.8, 2H), 1.64 (m, 2H), 0.80 (t, J = 7.6, 3H); ¹³C NMR (CD₃OD, 125 MHz) δ 189.0, 147.0, 142.9, 142.57, 136.9, 130.7, 127.5, 114.0, 48.9, 20.6, 9.7; HPLC (30–80%- Acetonitrile/H₂O-20min), T_R 4.98min, 98.1%. The sample was run on a Hypersil Gold C8 Column (5µm, 4.6 x 150mm) with solvent (30–80%- Acetonitrile/H₂O) gradually increasing the percentage of Acetonitrile from 30 to 80% over 20 mins.

Figure 4 IOI-214 prevents LPS-induced inflammation in murine macrophages and hepatocytes in culture.

Notes: (A) The murine macrophage cell line, RAW264.7 (A), and the murine hepatocyte cell line, AML-12 (B) were treated with 20 μM IOI-214 and DMSO (control) to determine if IOI-214 could block the upregulation of Tnf, Ifnb1, Ccl2, Il1b, and Il6 expression in the presence of 10 ng/mL LPS. All cells were pretreated with IOI-214 or DMSO for 1 hour prior to LPS treatment for 4 hours. Gene expression was determined via RT-qPCR. Error bars in all bar graphs represent ±SEM. Significance was determined using a one-way ANOVA followed by Tukey’s HSD post hoc analysis for multiple comparison. Bars indicate differences between indicated groups. ^#Indicates difference from all other groups, p < 0.05. *Indicates p < 0.05. ***Indicates p < 0.001. *****Indicates p < 0.00001.

Figure 5 IOI-214 blocks HF diet-induced mesenteric fat accumulation without affecting HF diet-induced weight gain or subcutaneous fat accumulation.

Notes: Seven-week-old male C57BL/6J mice were fed a HF diet and treated once daily with 1mg/kg IOI-214 or DMSO (10% in sterile saline) via intraperitoneal injection for 16 weeks. Weights of the mice were measured weekly and weight over the course of the experiment was plotted (A) and body weight gain was calculated (B). At the end of the 16-week experiment, mice were euthanized and mesenteric (C) and subcutaneous (D) fat was extracted from the mice and weighed. (B–D) Black squares (HF DMSO), gray triangles (HF IOI-214), and green diamonds (Chow Diet) represent data points from individual mice in each group. Error bars in all line and bar graphs represent ±SEM. Significance was determined using a one-way ANOVA followed by Tukey’s HSD post hoc analysis for multiple comparison. Bars in bar graphs indicate differences between indicated groups. Asterisks in the line graph indicate differences of HF DMSO and HF IOI-214 groups from the Chow Diet group at each time point. ***Indicates p < 0.001. *****Indicates p < 0.00001. ******Indicates p < 0.000001. Outliers are indicated by pink circles.

Figure 6 Continued.

Figure 6 IOI-214 ameliorates HF diet-induced hepatic steatosis.

Notes: Seven-week-old male C57BL/6J mice were fed a HF diet and treated once daily with 1mg/kg IOI-214 or DMSO (10% in sterile saline) via intraperitoneal injection for 16 weeks. (A) Oil Red O (ORO) staining and (B) Hematoxylin and eosin (H&E) staining was performed on liver tissue sections prepared from the mice after 16 weeks of HF diet feeding and treatment. All images in (A, top row) were taken at 40X magnification and images in (A, bottom row) were taken at 100X. All images in (B, top row) were taken at 20X and images in (B, bottom row) were taken at 200X. The black arrow in B represents an example of hepatocellular ballooning. Scale bars are as indicated in A&B. These are representative images from mice in each group. (C) Liver triglyceride content was quantified biochemically in liver tissue extracted from the mice after 16 weeks of treatment. (D) RNA was isolated from the liver of the HF IOI-214 and HF DMSO mice from the 16-week study and Plin5 gene expression was determined via RT-qPCR. (E) Pearson’s correlation (r) analysis of hepatic TG content and relative fold change in Plin5 expression in HF DMSO, HF IOI-214 and Chow Diet mice. (C–E) Black squares (HF DMSO), gray triangles (HF IOI-214), and green diamonds (Chow Diet) represent data points from individual mice in each group. Error bars in all bar graphs represent ±SEM. Significance was determined using a one-way ANOVA followed by Tukey’s HSD post hoc analysis for multiple comparison. Bars indicate differences between indicated groups. *Indicates p < 0.05. ****Indicates p < 0.0001. Outliers are indicated by pink circles.

Figure 7 IOI-214 mitigates HF diet-induced fasting hyperglycemia, without affecting fasting serum insulin, cholesterol or triglyceride levels, or glucose intolerance.

Notes: Blood was collected from the mice at the end of the 16-week study and fasting serum cholesterol (A), TG (B), insulin (C), and blood glucose (D) levels were measured. A 3-hour intraperitoneal glucose tolerance test (E) was performed at week 15. Black squares (HF DMSO), gray triangles (HF IOI-214), and green diamonds (Chow Diet) represent data points from individual mice in each group. Error bars in all line and bar graphs represent ±SEM. Significance was determined using a one-way ANOVA followed by Tukey’s HSD post hoc analysis for multiple comparison or Independent Samples t-tests as appropriate. Bars indicate differences between indicated groups in bar graphs. Asterisks in the line graph indicate differences of HF DMSO and/or HF IOI-214 groups from the Chow Diet group at each time point. *Indicates p < 0.05. **Indicates p < 0.01. ****Indicates p < 0.0001. Outliers are indicated by pink circles.

Figure 8 IOI-214 blocks HF diet-induced hepatic inflammatory cytokine and chemokine protein levels in vivo.

Notes: Protein levels of inflammatory cytokines [TNFα (A), IL6 (B) and IL1β(C)] and the chemokine MCP1 (D) were evaluated in the liver of the mice at the end of the 16-week experiment via cytokine and chemokine-specific ELISAs. Black squares (HF DMSO), gray triangles (HF IOI-214), and green diamonds (Chow Diet) represent data points from individual mice in each group. Error bars in all graphs represent ±SEM. Significance was determined using a one-way ANOVA followed by Tukey’s HSD post hoc analysis for multiple comparison. Bars indicate differences between indicated groups. *Indicates p < 0.05. **Indicates p < 0.01.

Figure 9 Correlations between hepatic TG content and inflammatory cytokine and chemokine protein levels in the liver of HF DMSO, HF IOI-214 and Chow Diet mice.

Notes: Pearson’s correlation (r) analyses of hepatic TG content and hepatic protein levels of TNFα (A), MCP1 (B), IL6 (C) and IL1β (D). Hepatic TG content was significantly and highly correlated with the hepatic protein levels of each of the cytokines and chemokine as indicated. The HF IOI-214 mice, in all cases, clearly had decreased levels of hepatic TG content compared to the HF DMSO mice that was correlated with decreased levels of the cytokines and chemokine evaluated.

Table 1 Effects of IOI-214 on Key Metabolites Related to Inflammation

	Liver		Serum
Relevant Physiological Process	Metabolite	Significant Fold Change in Metabolite Levels Between HF IOI-214 and HF DMSO Groups (Welch’s Two-Sample t-test)	Metabolite	Significant Fold Change in Metabolite Levels Between HF IOI-214 and HF DMSO Groups (Welch’s Two-Sample t-test)
Inflammation	Itaconate	2.38	Tryptophan	0.80
			Kynurenine	1.87

Note: Black arrows indicate significant increases or decreases in levels of the respective metabolites as indicated.

Figure 10 IOI-214 improves gut microbiota dysbiosis in HF diet-fed mice.

Notes: Fecal samples from the colon of HF IOI-214 and HF DMSO mice from the 16-week study were collected and sent to the UC Davis Mouse Metabolic Phenotyping Center (MMPC) for microbiome analysis. (A) The principal component analysis was generated using genus-level abundance data. (B) Phylum abundance levels were measured among fecal samples collected from HF-fed mice.

Figure 11 IOI-214 decreases gut permeability in HF diet-fed mice.

Notes: Serum samples from HF DMSO and HF IOI-214 collected at the end of the 16-week experiment and sent to the UC Davis Mouse Metabolic Phenotyping Center (MMPC) for analysis of LBP levels (A). (B) Pearson’s correlation (r) analysis of hepatic TG content and serum LBP levels in HF DMSO and HF IOI-214 mice. (C) Colon samples were collected from some of the mice at random at the end of the 16-week experiment, total protein was isolated, and Western blots were done to evaluate colonic OCLN protein levels. Densitometry was then conducted to evaluate the relative levels of OCLN protein relative to the amount of βActin. The average amount of OCLN per group was then calculated for the mice in each group wherein OCLN was present in the colon (n = 4 mice/group). Black squares (HF DMSO) and gray triangles (HF IOI-214) represent data points from individual mice in each group. Error bars represent ±SEM. Significance was determined using Independent Samples t-tests. Bars indicate differences between the groups. *Indicates p < 0.05. Outliers are indicated by pink circles.

Figure 12 Hypothesized working model of IOI-214 activity to ameliorate HF diet-induced NAFLD/MAFLD in vivo.

Notes: Green boxes represent IOI-214 targets in vivo. We present data in this manuscript that supports the hypothesis that IOI-214 ameliorates HF diet-induced NAFLD/MAFLD in vivo by improving gut microbiota dysbiosis (Figure 9), decreasing systemic inflammation (Table 1) and mesenteric fat accumulation (Figure 4C), decreasing gut permeability, and circulating levels of LPS (Figure 10), and decreasing hepatic inflammation (Figure 7). Thus, collectively, IOI-214 works at multiple levels in parallel to ameliorate the effects of the obesity-induced inflammation that drives NAFLD/MAFLD.