Malva parviflora extract ameliorates the deleterious effects of a high fat diet on the cognitive deficit in a mouse model of Alzheimer's disease by restoring microglial function via a PPAR-γ-dependent mechanism

Abstract

Background: Alzheimer's disease (AD) is a neuropathology strongly associated with the activation of inflammatory pathways. Accordingly, inflammation resulting from obesity exacerbates learning and memory deficits in humans and in animal models of AD. Consequently, the long-term use of non-steroidal anti-inflammatory agents diminishes the risk for developing AD, but the side effects produced by these drugs limit their prophylactic use. Thus, plants natural products have become an excellent option for modern therapeutics. Malva parviflora is a plant well known for its anti-inflammatory properties.

Methods: The present study was aimed to determine the anti-inflammatory potential of M. parviflora leaf hydroalcoholic extract (MpHE) on AD pathology in lean and obese transgenic 5XFAD mice, a model of familial AD. The inflammatory response and Amyloid β (Aβ) plaque load in lean and obese 5XFAD mice untreated or treated with MpHE was evaluated by immunolocalization (Iba-1 and GFAP) and RT-qPCR (TNF) assays and thioflavin-S staining, respectively. Spatial learning memory was assessed by the Morris Water Maze behavioral test. Microglia phagocytosis capacity was analyzed in vivo and by ex vivo and in vitro assays, and its activation by morphological changes (phalloidin staining) and expression of CD86, Mgl1, and TREM-2 by RT-qPCR. The mechanism triggered by the MpHE was characterized in microglia primary cultures and ex vivo assays by immunoblot (PPAR-γ) and RT-qPCR (CD36) and in vivo by flow cytometry, using GW9662 (PPAR-γ inhibitor) and pioglitazone (PPAR-γ agonist). The presence of bioactive compounds in the MpHE was determined by HPLC.

Results: MpHE efficiently reduced astrogliosis, the presence of insoluble Aβ peptides in the hippocampus and spatial learning impairments, of both, lean, and obese 5XFAD mice. This was accompanied by microglial cells accumulation around Aβ plaques in the cortex and the hippocampus and decreased expression of M1 inflammatory markers. Consistent with the fact that the MpHE rescued microglia phagocytic capacity via a PPAR-γ/CD36-dependent mechanism, the MpHE possess oleanolic acid and scopoletin as active phytochemicals.

Conclusions: M. parviflora suppresses neuroinflammation by inhibiting microglia pro-inflammatory M1 phenotype and promoting microglia phagocytosis. Therefore, M. parviflora phytochemicals represent an alternative to prevent cognitive impairment associated with a metabolic disorder as well as an effective prophylactic candidate for AD progression.

Keywords: Alzheimer’s Disease; Malva parviflora; PPAR-γ/CD36; inflammation; microglia; obesity.

Fig. 1

The Malva parviflora hydroalcoholic extract inhibits LPS-induced NF-κB activity in mouse RAW-Blue macrophages. a RAW-Blue macrophages were untreated or treated with LPS (100 ng/mL) in the presence of the indicated concentrations of M. parviflora hydroalcoholic extract (HE) and 12 h later embryonic alkaline phosphatase (SEAP) activity (driven by NF-κB/AP-1 activation) was determined in the supernatants as described in the “Methods” section. Values are expressed as fold increase relative to SEAP reporter activity in untreated control cells. b RAW-Blue macrophages were pre-treated with LPS or M. parviflora for 30 min and 12 h later embryonic alkaline phosphatase (SEAP) activity (driven by NF-κB/AP-1 activation) was determined in the supernatants as described in the “Methods” section. Data are shown as mean ± SEM. Statistical analysis was performed by two-way ANOVA with repeated measures followed by post hoc Sidak’s multiple comparisons test. This analysis revealed a significant effect for the MpHE concentration F(3,12) = 28.9, p < 0.001; for the LPS administration F(1,4) = 31.49 p = 0.005 and for the M. parviflora and LPS interaction F(3,12) = 30.88, p < 0.001. (***p < 0.001, *p = 0.03). c Cellular viability of RAW-Blue macrophages was evaluated using trypan blue exclusion after exposure to the MpHE at three different concentrations (0.1, 0.5, and 1.0 mg/mL) for 12 h in presence or absence of LPS (100 ng/mL). d RAW-Blue cells were treated with the indicated amount of the M. parviflora in the absence (Ctrl) or in the presence of LPS (100 ng/mL) for 12 h. Cells were fixed and stained with phalloidin and DAPI for F-actin (green) and nuclei (blue) detection, respectively. Images were captured using the Olympus FluoView 1000 confocal multiphoton microscope (Scale bar, 30 μm)

Fig. 2

Malva parviflora hydroalcoholic extract promotes insulin sensitivity and glucose tolerance in 5XFAD transgenic mice fed with high-fat diet. a Graphical time line of the study design and experimental procedures. GTT, glucose tolerance test; IRT, insulin resistant test; IFC, immunofluorescence. b Body weight gain (g) was measured weekly during 28 weeks in wild type (Wt) mice fed with normal diet (ND; closed black circle) or high-fat diet (HFD; closed black triangle) and those that received intragastrically water or 50 mg/kg/day of the MpHE (Mp): Wt ND + Mp (open black circle) or Wt HFD + Mp (open black triangle), or transgenic 5XFAD mice fed with a normal diet (ND; closed red circle) or a high-fat diet (HFD; closed red triangle) that received intragastrically water or 50 mg/kg/day of the MpHE (Mp): 5XFAD ND + Mp (open red circle), or 5XFAD HFD + Mp (open red triangle). Data are shown as mean ± SEM, n = 4 in Wt ND, n = 4 in Wt HFD, n = 4 in Wt ND + Mp, n = 4 in Wt HFD + Mp, n = 10 in 5XFAD ND, n = 10 in 5XFAD HDF, n = 10 in 5XFAD ND + Mp and n = 9 in 5XFAD HFD + Mp. Statistical analysis was performed by two-way ANOVA with repeated measures followed by post hoc Bonferroni’s multiple comparisons test. This analysis revealed a significant effect for the genotype F(7,47) = 9.971, p < 0.001; for the time F(27,1269) = 206.5 p < 0.001 and for the genotype and time interaction F(189, 1269), p < 0.001. (*p = 0.0488, **p = 0.0069, ***p < 0.001). c Basal blood glucose concentrations in Wt or transgenic 5XFAD mice after 8 months of being fed with ND or HFD alone or treated with Mp. Data are shown as mean ± SEM, n = 4 for all the groups. Statistical analysis was performed by three-way ANOVA followed by Tukey’s multiple comparisons test. This analysis revealed a significant effect for the genotype F(1,24) = 5.029, p = 0.03440; for the diet F(1,24) = 25.08, p < 0.001; for the M. parviflora treatment F(1,24) = 18.56; p = 0.002; for the genotype and diet interaction F(1,24) = 11, p = 0.0029; for the M. parviflora treatment and diet interaction F(1,24) = 24.99, p < 0.001; for the genotype and M. parviflora treatment interaction F(1,24) = 8.661 p = 0.0071; and for the genotype, M. parviflora treatment, and diet interaction F(1,24) = 0.2458, p = 0.6245. d Blood glucose during intraperitoneal glucose tolerance test (GTT) of Wt or transgenic 5XFAD mice 8 months after of being fed with ND or HFD alone or treated with M. parviflora (Mp). Bar graph (lower panel) represents the area under the curve (AUC). Data are shown as mean ± SEM, n = 4 for all the groups. Statistical analysis was performed by three-way ANOVA followed by Tukey’s multiple comparisons test. This analysis revealed a significant effect for the genotype F(1,24) = 36.52, p < 0.001; for the diet F(1,24) = 45.37, p < 0.001; for the M. parviflora treatment F(1,24) = 46.23, p < 0.001; for the genotype and diet interaction F(1,24) = 0.0002756, p = 0.9869; for the M. parviflora treatment and diet interaction F(1,24) = 9.277, p = 0.0056; for the genotype and M. parviflora treatment interaction F(1,24) = 3.678 p = 0.0671; and for the genotype, M. parviflora treatment, and diet interaction F(1,24) = 0.5456, p = 0.4673. e Blood glucose levels were measured at several time points following insulin administration during the insulin resistance test (IRT) of Wt or transgenic 5XFAD mice 8 months after of being fed with ND or HFD alone or treated with M. parviflora (Mp). Bar graph (lower panel) represents the area under the curve (AUC). Statistical analysis was performed by three-way ANOVA followed by Tukey’s multiple comparisons test. This analysis revealed a significant effect for the genotype F(1,24) = 356.6, p < 0.001; for the diet F(1,24) = 38.47, p < 0.001; for the M. parviflora treatment F(1,24) = 36.14, p < 0.001; for the genotype and diet interaction F(1,24) = 7.772, p = 0.0102; for the M. parviflora treatment and diet interaction F(1,24) = 4.078, p = 0.0547; for the genotype and M. parviflora treatment interaction F(1,24) = 15.14 p = 0.007; and for the genotype, M. parviflora treatment, and diet interaction F(1,24) = 3.468, p = 0.0749

Fig. 3

Malva parviflora hydroalcoholic extract reduces amyloid plaque formation and astrogliosis in 5XFAD transgenic mice fed with high-fat diet. a (Left) Representative micrographs of amyloid plaques labeled with thioflavin S in the hippocampus of wild type (Wt) or 5XFAD mice fed with either normal diet (ND) or high-fat diet (HFD) non-treated (Vehicle) or treated with Malva parviflora hydroalcoholic extract (M. parviflora) for 8 months (scale bar 100 μm). (Right) Graph represents amyloid plaque loads in hippocampus of 5XFAD transgenic mice fed with normal diet (ND) or high-fat diet (HFD), treated with vehicle or M. parviflora extract. Data are shown as mean ± SEM, n = 4 animals per group. Statistical analysis was performed by three-way ANOVA followed by Tukey’s multiple comparisons test. This analysis revealed a significant effect for the genotype F(1,24) = 113.7, p < 0.001; for the diet F(1,24) = 7.781, p = 0.01; for the M. parviflora treatment F(1,24) = 39.54, p < 0.001; for the genotype and diet interaction F(1,24) = 7.781, p = 0.01; for the M. parviflora treatment and diet interaction F(1,24) = 3.03 p = 0.094; for the genotype and M. parviflora treatment interaction F(1,24) = 39.54 p < 0.001; and for the genotype, M. parviflora treatment, and diet interaction F(1,24) = 3.03, p = 0.094. b Astrogliosis was evaluated by immunofluorescence on hippocampal slides (dentate gyrus) from Wt and 5XFAD mice fed with high-fat diet (HFD) non-treated (vehicle) or treated with M. parviflora extract for 8 months. Astrocytes were imaged using the Olympus FluoView 1000 confocal multiphoton microscope (× 40, scale bar 30 μm). Lower row represents a × 2 digital magnification from boxed areas (scale bar 30 μm). Sketch indicates the analyzed area within the hippocampus

Fig. 4

Malva parviflora hydroalcoholic extract protects 5XFAD transgenic mice from memory deficit. Morris Water Maze test was performed to evaluate spatial memory in 5XFAD mice treated with Malva parviflora hydroalcoholic extract (M. parviflora). a Time (sec) need to reach the hidden platform (escape latency) during the five acquisition days (test trial) of wild type (Wt) mice fed with normal diet (ND) (closed black cirle) or high-fat diet (HFD) (closed black triangle) and those that received intragastrically water or 50 mg/kg/day of the MpHE (Mp): Wt ND + Mp (open black circle) or Wt HFD + Mp (open black triangle), or transgenic 5XFAD mice fed with a normal diet (ND) (closed red circle) or a high-fat diet (HFD) (closed red triangle) that received intragastrically water or 50 mg/kg/day of the MpHE (Mp): 5XFAD ND + Mp (open red circle), or 5XFAD HFD + Mp (open red triangle). Data are shown as mean ± SEM, n = 8 animals per group. Statistical analysis was performed by two-way ANOVA followed by Sidak’s multiple comparisons test. This revealed a significant effect for time F(4,108) = 12.16, p < 0.0001, Genotype F(7,27) = 11.17, p < 0.0001 and for the interaction between time and genotype F(28,108) = 1.969, p = 0.007 (day 4 Wt ND vs 5XFAD HFD *p = 0.04; day 5 Wt ND vs 5XFAD HFD ***p < 0.0001). b Area under the curve (AUC) of the latencies for each group was calculated using the trapezoidal rule. Data are shown as mean ± SEM, n = 8 animals per group. Statistical analysis was performed by three-way ANOVA followed by Tukey’s multiple comparisons test. This analysis revealed a significant effect for the genotype F(1,56) = 14.95, p < 0.001; for the M. parviflora treatment F(1,56) = 15.69, p < 0.001; for the genotype and diet interaction F(1,56) = 1.401, p = 0.24; for the M. parviflora treatment and diet interaction F(1,56) = 5.734, p = 0.02; for the genotype and M. parviflora treatment interaction F(1,56) = 2.328, p = 0.13; for the genotype, M. parviflora treatment and diet interaction F(1,56) = 2.467, p = 0.12. c Time (seconds; sec) to platform for each group during the probe trial (day eight) in the absence of platform. Statistical analysis was performed by three-way ANOVA followed by Tukey’s multiple comparisons test. This analysis revealed a significant effect for the genotype F(1,56) = 15.46, p < 0.001; for the M. parviflora treatment F(1,56) = 25.97, p < 0.001; for diet F(1,56) = 0.5947, p = 0.44; for the genotype and diet interaction F(1,56) = 5.703, p = 0.02; for the M. parviflora treatment and diet interaction F(1,56) = 1.915, p = 0.17; for the genotype and M. parviflora treatment interaction F(1,56) = 5.131, p = 0.03; for the genotype, M. parviflora treatment and diet interaction F(1,56) = 0.04011, p = 0.84. d Representative swimming paths of mice during the probe trial on day eight are depicted. The hidden platform was located on the NW quadrant

Fig. 5

Malva parviflora hydroalcoholic extract regulates LPS-induced microglia activation. Microglia primary cultures were isolated from neonatal wild type animals as described in the “Methods” section. Microglia enrichment was determined as follows. a Confocal microscopy was used to examine GFAP (astrocytes) and F4/80 (microglia) expression in mixed cultures and after microglia purification (Microglia). Nuclei were visualized by DAPI staining (scale bar, 30 μm). b Microglia enrichment was determined by flow citometry using anti-CD11b antibodies. c The mRNA levels of different cell markers, microglia (F4/80); astrocytes (GFAP) and neurons (Neurofilament M; NF) were determined in the mixed cultures (MC), isolated microglia (microglia), whole brain (WB) and CHO cells by RT-PCR analysis as described in the “Methods” section. Actin levels were used as internal control. d Microglial cultures were exposed to PBS (Ctrl), LPS (100 ng/mL), MpHE (M. parviflora) (1 mg/mL) or LPS (100 ng/mL) and MpHE (1 mg/mL) (LPS + M. parviflora) for 24 h. Cells were fixed, stained with phalloidin and DAPI for F-actin (green) and nuclei (blue) detection, respectively. Microglia was imaged using the Olympus FluoView 1000 confocal multiphoton microscope (scale bar, 30 μm). The boxed areas were × 2 digitally magnified and shown as inset. e The microglia morphologies were classified as ramified, amoeboid and intermediate shapes in the different groups described in (D); MpHE (Mp). One hundred cells were measured for each experimental condition. Data (mean ± SEM) were analyzed by one-way ANOVA followed by Tukey’s post hoc test (^**p < 0.01; ^***p < 0.001). f Microglial cultures were exposed to PBS (Control) or LPS (100 ng/mL) in the presence or absence Vehicle) of MpHE (1 mg/mL) (Mp) for 24 h. The length of the protrusions (μm) of the microglia were determined using Neurite Tracer from ImageJ software (National Institutes of Health, Bethesda, MD). Data (mean ± SEM) were analyzed by two-way ANOVA followed by post hoc Bonferroni’s multiple comparisons test. This analysis revealed a significant effect for the LPS F(1,8) = 13.32, p = 0.006; not for the treatment with M. parviflora F(1,8) = 0.8054 p = 0.40 or for the LPS and M. parviflora interaction F(1,8) = 1.245 p = 0.30. g Microglial cultures were exposed to PBS (Control), MpHE (1 mg/mL) (Mp) or LPS (100 ng/mL) and MpHE (1 mg/mL) (LPS + Mp) for 24 h. The phagocytic index was calculated by multiplying the percentage of microglia with internalized latex beads by the average number of internalized latex beads per each group. Data were collected from seven random fields per group and analyzed by one-way ANOVA followed by Tukey’s post hoc test (^***p < 0.001 vs control)

Fig. 6

Ex vivo treatment of microglial cells from 5XFAD mice with Malva parviflora hydroalcoholic extract increases the microglia phagocytic activity. a Microglia was purified from adult (wild type and 5XFAD) mouse brains as described in the “Methods” section. Microglia enrichment was determined by flow citometry using anti-CD11b antibodies. A representative histogram depicts isotype (red) and CD11b (blue) labeled cells from 5XFAD transgenic brain. b Primary microglial cells were purified from 8- and 10-months-old Wt and 5XFAD mice as described in the “Methods” section and left untreated (Ctrl) or treated with the M. parviflora extract (1 mg/mL) for 24 h and then exposed to fluorescent E. coli (red) for 4 h. Cells were fixed and stained with phalloidin and DAPI for F-actin (green) and nuclei (blue) detection, respectively. Microglia was imaged using the Olympus FluoView 1000 confocal multiphoton microscope (Scale bar, 30 μm). Insets show a × 2 digital magnification from boxed areas. c Graph depicts the percentage of cells with internalized E. coli (m.o.) that were left untreated (−) or treated (+) with the M. parviflora extract (1 mg/mL) for 24 h and then exposed to fluorescent E. coli (red) for 4 h. Data (mean ± SEM) were analyzed by one-way ANOVA followed by Tukey’s post hoc test (^*p < 0.05). d Average of microorganisms (E. coli) internalized by microglia from the indicated mouse strain and age untreated (−) or treated (+) with the M. parviflora extract (intervals were from 1 to 5, from 6 to 10 and greater than 10 microorganisms/cell). Data (mean ± SEM) were analyzed by paired t test (^**p < 0.01; ^***p < 0.001). e Phagocytic index was calculated by multiplying the percentage of microglia with internalized bacteria by the average number of internalized E. coli bacteria per each group. Data were collected from seven random fields per group and analyzed by one-way ANOVA followed by Tukey’s post hoc test (^***p < 0.001)

Fig. 7

Malva parviflora hydroalcoholic extract increases microglia accumulation around the Aβ plaques in the cortex and hippocampus of 5XFAD mice. Amyloid plaques were labeled with thioflavin S (green) and microglia with anti-Iba-1 antibodies (red) on cortical a or hippocampal sections (dentate gyrus) b from 5XFAD mice fed with either normal diet (ND) or high-fat diet (HFD) non-treated (Vehicle) or treated with M. parviflora extract (M. parviflora) for 8 months. Representative micrographs are depicted. Images were captured using the Zeiss Axioskop Observer Z1 inverted fluorescence microscope (× 10, scale bar 100 μm). Lower row represents a × 3 digital magnification from boxed areas (scale bar 30 μm). Sketch indicates the analyzed area within the cortex and the hippocampus, respectively

Fig. 8

Malva parviflora hydroalcoholic extract attenuates microglia pro-inflammatory M1 phenotype in the cortex of 5XFAD mice. Total RNA was isolated from the cortex of Wt or 5XFAD mice fed with either normal diet (ND) or high-fat diet (HFD) non-treated (Vehicle) or treated with MpHE (M. parviflora) for 8 months. a The transcript levels of CD86 (marker of M1 state) were determined by RT-qPCR as described in the “Methods” section. Data are shown as mean ± SEM, n = 3 animals per group. Statistical analysis was performed by three-way ANOVA followed by Tukey’s multiple comparisons test. This analysis revealed a significant effect for the genotype F(1,16) = 42.45, p < 0.001; for the diet F(1,16) = 0.4022, p = 0.53; for the M. parviflora treatment F(1,16) = 29.79, p < 0.001; for the genotype and diet interaction F(1,16) = 0.04041, p = 0.84; for the M. parviflora treatment and diet interaction F(1,16) = 0.2594 p = 0.62; for the genotype and M. parviflora treatment interaction F(1,16) = 20.67 p < 0.001; for the genotype, M. parviflora treatment and diet interaction F(1,16) = 2.037, p = 0.17. b TNF (marker of M1 state) mRNA levels. Data are shown as mean ± SEM, n = 3 animals per group. Statistical analysis was performed by three-way ANOVA followed by Tukey’s multiple comparisons test. This analysis revealed a significant effect for the genotype F(1,16) = 25.65 p < 0.001; for the diet F(1,16) = 5.758, p = 0.03; for the M. parviflora treatment F(1,16) = 32.4, p < 0.001; for the genotype and diet interaction F(1,16) = 4.955, p = 0.04; for the M. parviflora treatment and diet interaction F(1,16) = 2.259 p = 0.15; for the genotype and M. parviflora treatment interaction F(1,16) = 26.77 p < 0.001; for the genotype, M. parviflora treatment and diet interaction F(1,16) = 2.189, p = 0.16. c Mgl1 (marker of M2 state) mRNA levels. Data are shown as mean ± SEM, n = 3 animals per group. Statistical analysis was performed by three-way ANOVA followed by Tukey’s multiple comparisons test, and d TREM-2 mRNA levels. Data are shown as mean ± SEM, n = 3 animals per group. Statistical analysis was performed by three-way ANOVA followed by Tukey’s multiple comparisons test. Microglia from 8-month-old Wt or 5XFAD mice were unstimulated or stimulated with LPS (100 ng/mL) in the presence or absence of MpHE (M. parviflora; 1 mg/mL) for 24 h. Control cells were treated with PBS (Ctrl) or MpHE alone (M. parviflora). Supernatants were used to determine TNF and IL6 levels by ELISA as described in the “Methods” section. e TNF levels. Data are shown as mean ± SEM, n = 3 animals per group. Statistical analysis was performed by three-way ANOVA followed by Tukey’s multiple comparisons test. This analysis revealed a significant effect for the genotype F(1,16) = 7.878, p = 0.0127; for the LPS treatment F(1,16) = 17.74, p = 0.0007; for the M. parviflora treatment F(1,16) = 66.30, p < 0.0001; for the genotype and LPS treatment interaction F(1,16) = 6.105, p = 0.0251. f IL6 levels. Data are shown as mean ± SEM, n = 3 animals per group. Statistical analysis was performed by three-way ANOVA followed by Tukey’s multiple comparisons test. This analysis revealed a significant effect for the genotype F(1,16) = 25.76, p = 0.0001; for the LPS treatment F(1,16) = 19.86, p = 0.0004; for the M. parviflora treatment F(1,16) = 309.3, p < 0.0001; for the genotype and LPS treatment interaction F(1,16) = 20.71, p = 0.0003

Fig. 9

The Malva parviflora hydroalcoholic extract contains scopoletin and oleanolic acid. a HPLC chromatograms comparing the hydroalcoholic (HA) and ethyl acetate (AcOEt) fractions from MpHE with reference standards of scopoletin and oleanolic acid. All samples were monitored at 345 nm. b Chemical structure of scopoletin. c Chemical structure of oleanolic acid. d Principal compounds present in the hydroalcoholic extract of M. parviflora

Fig. 10

Malva parviflora hydroalcoholic extract regulates the phagocytic capacity of microglial cells via PPARγ-CD36 mediated mechanism. Microglial primary cultures were left untreated or pre-treated with GW9662, a specific PPAR-γ inhibitor for 1 h, following by incubation with MpHE or the PPAR-γ agonist, pioglitazone, at the indicated concentration for 24 h. a The PPAR-γ levels from the cell extracts of microglial primary cultures were determined by immunoblot using specific antibodies and the actin levels were used as internal control. Normalized densitometry values of PPAR-γ (PPAR-γ/actin) present in the extracts of microglial primary cultures in the presence of MpHE (Mp) or pioglitazone and GW9662. Data are shown as mean ± SEM and were analyzed by one-way ANOVA. Ctrl vs Mp 0.1 mg/mL (**p = 0.009); Ctrl vs Mp 1 mg/mL (**p = 0.004); Ctrl vs pioglitazone (**p = 0.001); Mp 0.1 mg/mL vs GW9662 + Mp 0.1 mg/mL (*p = 0.04); Mp 1 mg/mL vs GW9662 + Mp 1 mg/mL (**p = 0.003); Pioglitazone vs GW9662 + Pioglitazone (***p < 0.001). b CD36 mRNA levels were determined by qPCR using total RNA from the microglial primary cultures treated as described above. Data were analyzed by one-way ANOVA. Ctrl vs Mp (*p = 0.04); Ctrl vs pioglitazone (**p = 0.002); Mp vs GW9662 + Mp (*p = 0.04); pioglitazone vs GW9662 + pioglitazone (**p = 0.005). c Representative micrographs from cortex of adult 5XFAD mice alone (Bs) or in the presence of microglial primary cultures (5XFAD Bs + microglia) untreated (−) or pre-treated with M. parviflora or pioglitazone and GW9662. The slices were stained with thioflavin S and analyzed by confocal microscopy. d The plaques number were quantified. Data were analyzed by one-way ANOVA. Bs vs M. parviflora (***p < 0.001); Bs vs pioglitazone (*p = 0.03); M. parviflora vs GW9662 + M. parviflora (*p = 0.04). e The plaques size was quantified. Data were analyzed by one-way ANOVA. Bs vs M. parviflora (*p = 0.03); Ctrl vs M. parviflora (**p = 0.007); M. parviflora vs GW9662 + M. parviflora (*p = 0.02)

Fig. 11

Malva parviflora hydroalcoholic extract regulates the phagocytic capacity of microglial cells via PPARγ-CD36 mediated mechanism in the 5xFAD transgenic mice. a Graphical time line of the study design and experimental procedures. The 5XFAD transgenic mice received 50 mg/kg/day of the MpHE (Mp) or water (Vehicle) intragastrically during 2 months. After, the mice were intraperitoneally (IP) injected with GW9662 (5 mg/kg), a specific PPAR-γ inhibitor, or vehicle (5% DMSO/95%PBS) for the last 3 days before sacrifice. For the in vivo amyloid β phagocytosis assay, mice were intraperitoneally injected 6 h before sacrifice with methoxy-X04 (10 mg/kg). The presence of Aβ peptides in the microglial cells were analyzed by flow cytometry. b CD36 expression in microglia (CD11b⁺) from 5XFAD transgenic mice untreated (Vehicle) or treated with MpHE alone (M. parviflora) or with MpHE and GW9662 (M. parviflora+GW9662) determined by flow cytometry. Left panel: A representative histogram depicts CD36 expression in the CD11b⁺ cells from 5XFAD transgenic brain. Middle panel: % of the CD11b⁺/CD36⁺ cells. Data (mean ± SD) were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Percentage CD11b+/CD36+ 5XFAD vehicle vs 5XFAD Mp (*p = 0.0298); 5XFAD Mp vs 5XFAD Mp + GW9662 (*p = 0.0291). Right panel: CD36 expression levels. MFI CD11b⁺/CD36⁺ 5XFAD Vehicle vs 5XFAD Mp (*p = 0.0228). c Left panel: cytometry analysis of Aβ peptides phagocytized by microglia (CD11b+/CD36+) from adult 5XFAD mice untreated (Vehicle) alone (M. parviflora) or with MpHE and GW9662 (M. parviflora+GW9662) that received methoxy-X04 (M-X04). Left panel: representative histogram is shown. Middle panel: percent of the CD36⁺/M-X04⁺ cells data (mean ± SD) were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Percentage CD36⁺/Methoxy-X04⁺ 5XFAD vehicle vs 5XFAD Mp (**p = 0.0089); 5XFAD Mp vs 5XFAD Mp + GW9662 (**p = 0.0069). Right panel: phagocyted M-XO4 levels. MFI CD36⁺/Methoxy-X04 5XFAD Vehicle vs 5XFAD Mp (**p = 0.0047); 5XFAD Mp vs. 5XFAD Mp + GW9662 (**p = 0.0054)

Fig. 12

Malva parviflora hydroalcoholic extract regulates PPARγ levels in the 5XFAD transgenic mice. The 5XFAD and Wt mice were fed with either normal diet (ND) or high-fat diet (HFD) non-treated (Vehicle) or treated with M. parviflora hydroalcoholic extract (M. parviflora). The PPAR-γ levels from the frontal cortex cell extracts were determined by immunoblot using specific antibodies and the GAPDH levels were used as internal control. a Normalized densitometry values of PPAR-γ (PPAR-γ/GAPDH) present in the frontal cortex cell extracts of 5XFAD and Wt mice. Data (mean ± SD) were analyzed by three-way ANOVA followed by Tukey’s multiple comparisons test. This analysis revealed a significant effect for the genotype F(1,16) = 7.529, p = 0.0144; for the diet F(1,16) = 13.37, p = 0-0021; for the M. parviflora treatment F(1,16) = 28.11, p < 0.0001; for the genotype and diet interaction F(1,16) = 0.5415, p = 0.4725; for the M. parviflora treatment and diet interaction F(1,16) = 17.37, p = 0.0007; for the genotype and M. parviflora treatment interaction F(1,16) = 6.946, p < 0.0180; for the genotype, M. parviflora treatment and diet interaction F(1,16) = 6.111, p = 0.0250. b Microglial primary cultures were left untreated or pre-treated with GW9662, a specific PPAR-γ inhibitor for 1 h, following by incubation with oleanolic acid (OA), at the indicated concentration for 24 h. The PPAR-γ levels from the cell extracts of microglial primary cultures were determined by immunoblot using specific antibodies and the GAPDH levels were used as internal control. Normalized densitometry values of PPAR- γ (PPAR- γ/GAPDH) present in the extracts of microglial primary cultures in the presence of oleanolic acid. Data (mean ± SD) were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Ctrl vs 4.5 μg/mL oleanolic acid (*p = 0.0414); 4.5 μg/mL oleanolic acid vs GW9662 + oleanolic acid 4.5 μg/mL (*p = 0.0116); 45 μg/mL oleanolic acid vs GW9662 + 45 μg/mL oleanolic acid (**p = 0.0079); c Model of the mechanism by which M. parviflora hydroalcoholic extract (HE) diminishes neuroinflammation. Chronic inflammation compromises microglia clearance functions by reducing the expression of the scavenger receptor CD36. The peroxisome proliferator-activated receptor (PPAR-γ) suppress inflammatory gene expression and promotes phagocytosis by regulating CD36 expression a scavenger receptor involved in microglia-dependent amyloid plaque destruction. According with this, our results indicate that a component present in the M. parviflora extract, probably oleanolic acid, based on previous studies [43] and our data, induces PPAR-γ activation that results in the upregulation of the scavenger receptor CD36 expression, thus leading to microglia-enhanced phagocytic, amyloid plaque clearance activity, diminished neuroinflammation, and improved learning and memory