Jinmaitong ameliorates diabetic peripheral neuropathy in streptozotocin-induced diabetic rats by modulating gut microbiota and neuregulin 1

JMT alleviated peripheral neuropathy phenotypes in DPN rats

Twelve weeks after the induction of diabetes, STZ-injected rats displayed the peripheral neuropathy phenotypes characterized by increased non-fasting blood glucose level (Figure 1A, p<0.0001), decreased body weight (Figure 1B, p<0.0001), reduced Mechanical Withdraw Threshold (MWT) (Figure 1C, p<0.0001), and loss of intraepidermal nerve fibers in the hind paw (Figure 1D, E, p<0.0001). Both JMT and NTP treatment increased MWT (Figure 1C, p<0.0001) without affecting non-fasting blood glucose level and body weight in DPN rats (Figure 1A, 1B, p>0.05). In addition, JMT, but not NTP, improved intraepidermal nerve fiber density (IENFD) in DPN rats (p<0.05) compared to distilled water-treated DPN rats (Figure 1D, 1E, p>0.05). In summary, both JMT and NTP alleviated some peripheral neuropathy symptoms in DPN rats, with NTP showing no predominance than JMT.

Figure 1. Effects of JMT on phenotypes and histological morphology in DPN rats after 12 weeks. (A) Non-fasting blood glucose level, (B) body weight, (C) mechanical withdraw threshold, (D) quantitation of intraepidermal nerve fiber density (IENFD) in different groups. (E) Representative images of PGP 9.5 (green) immunostaining of intraepidermal nerve fiber profiles at a magnification of 200×; scale bars, 100 μm. Representative HE staining photomicrographs of (F) cross-sections and (G) longitudinal sections from normal control rats, distilled water-treated DPN rats, JMT-treated DPN rats, and NTP-treated DPN rats at a magnification of 400×; scale bars, 50 μm. Means ± SD; n=3-8/group. ****p <0.0001 vs. normal control group; ††††p <0.0001, †p <0.05 vs. distilled water-treated DPN group. One-way ANOVA followed by Tukey’s multiple comparisons test or Kruskal-Wallis test followed by Dunn’s multiple comparisons test.

JMT improved the neurological morphology of sciatic nerves in DPN rats

Both hematoxylin and eosin (HE) staining (Figure 1F, 1G) and ultrastructural evaluation using a transmission electron microscope (TEM) (Figure 2A, 2B) showed that sciatic nerves from normal control rats contained myelinated fibers of varying diameters, regular contours, intact myelin sheaths, and thickness proportional to the diameter of their axons, which were uniformly distributed. In the distilled water-treated DPN group, myelinated fibers with axonal atrophy and myelin sheath deformation were observed and were sparsely distributed under the light microscopy (Figure 1F, 1G). Meanwhile, ultrastructure examinations revealed that myelin destruction had occurred as evidenced by onion-bulb and bubble form protrusions on the myelin sheath and axolemma border of myelinated axons. The most striking ultrastructural alterations of axonal myelin were vacuolization and lamellar separation, which consists of separated myelin fibers with large spaces between the axon and myelin sheath. Shrunken and swollen axons were also commonly observed, and both total damage and axonal deformations were present (Figure 2A, 2B). Qualitative assessment of ultrastructural characteristics revealed that the number of myelinated axons per 5000 μm² (Figure 2C) decreased, while the percentage of abnormal myelin fibers with morphological alterations, including myelin infoldings, vacuolization, and uneven thickness increased in distilled water-treated DPN rats (Figure 2D).

Figure 2. Ultrastructure and morphometry of sciatic nerves under transmission electron microscopy. Representative images of (A) cross-sections and (B) longitudinal sections of sciatic nerves from different groups at magnifications of 8000×, 20000×, and 80000×; scale bars, 2 μm, 500 nm, and 200 nm, respectively. Morphometric analyses of sciatic nerve showing (C) the number of myelinated axons per 5,000 μm² and (D) the percentage of abnormal myelin fibers in different groups. Means ± SD; n = 3/group. (E) Quantification of g ratios for axons in different groups. n=632 axons from 3 normal control rats, n=525 axons from 3 distilled water-treated DPN rats, n=585 axons from JMT-treated DPN rats, n=544 axons from 3 NTP-treated DPN rats. ***p < 0.001, *p < 0.05, vs. normal control group; †††p < 0.001, †p < 0.05 vs. distilled water-treated DPN group; ‡‡‡p < 0.001 vs. JMT-treated DPN group. One-way ANOVA followed by Tukey’s multiple comparison test or Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Pound signs (#) indicate onion-bulb and bubble form protrusions. Red arrows indicate lamellar separation between the axon and myelin sheath and demyelination.

Morphological abnormalities were ameliorated in both JMT-treated and NTP-treated DPN rats. The sciatic nerves of JMT-treated DPN rats contained myelin fibers of various sizes and with more proportional caliber sheaths surrounding the axon. Under light microscopy, myelin sheaths showed more regular contours with homogeneous distribution in JMT- and NTP-treated DPN rats compared with the distilled water-treated DPN rats (Figure 1F, 1G). JMT and NTP improved pathological ultrastructure features in myelin and axons to different degrees. Vacuolization and lamellar separation of axonal myelin were reduced to a greater extent after JMT treatment. Morphologic alterations and myelin breakdown were also reduced specifically in JMT-treated DPN rats, and deranged myelin sheath cells recovered after treatment (Figure 2A, 2B). Qualitative assessment of ultrastructural characteristics revealed that the number of myelinated axons per 5000 μm² increased (Figure 2C, p < 0.05) and the percentage of abnormal myelin fibers decreased (Figure 2D, p < 0.001) in JMT-treated DPN rats, but not in NTP-treated DPN rats (Figure 2C, 2D, p > 0.05), compared to distilled water-treated DPN rats. Axons g ratios did not differ among different groups (Figure 2E, p > 0.05) [24], indicating that atrophy of myelinated fibers occurred to the same degree in both axons and myelin sheaths [25]. The observed g ratio around 0.6 may explain, at least in part, the preservation of baroreceptor function in the DPN rats [26].

JMT prevented myelin and axonal damage and increased serum neuregulin 1 (NRG1) level in DPN rats

Demyelination and axonal degeneration are two key pathologic features of DPN. Myelin pathology is usually measured by paranodal architecture, while axonal damage is usually evaluated by measuring acute axonal damage indicator amyloid precursor protein (APP) levels [27, 28].

Immunolabeling for contactin-associated protein (Caspr) at the paranodes is often used to quantify symmetrical paranodal densities and asymmetrical heminode densities, which are key indicators of structural damage and paranodal demyelination in peripheral nerves [29]. Here, immunostaining revealed that the number (Figure 3A, 3D, p < 0.001) and percentage (Figure 3A, 3B, p < 0.01) of Caspr-expressing paranodes were decreased, while heminode percentage was increased (Figure 3A, 3C, p < 0.01) in the sciatic nerves of distilled water-treated DPN rats compared to normal control rats. These changes were reversed by JMT treatment (Figure 3A–3D, p < 0.05), further suggesting that JMT protects myelin and paranodal architecture in DPN rats. In contrast, no differences were observed in these measures between NTP-treated and distilled water-treated DPN rats (Figure 3A–3D, p > 0.05). No significant differences in number of heminodes were observed among all the different groups (Figure 3E, p > 0.05), perhaps indicating that symmetrical paranodes were more severely damaged than asymmetrical heminodes in DPN rats.

Figure 3. JMT protected paranodal structures in the sciatic nerves of DPN rats. (A) Representative images of longitudinal sections of sciatic nerves with immunolabeling for Caspr (pink) and counterstained with DAPI (blue, marking nuclei) at a magnification of 400×; scale bars, 20 μm. Percentage of (B) paranodes and (C) heminodes at paranodal junctions in the sciatic nerves of rats. Number of (D) paranodes and (E) heminodes per mm² in the sciatic nerves of rats. Means ± SD; n=3/group. ***p < 0.001, **p < 0.01 vs. normal control group; ††P < 0.01, †P < 0.05 vs. distilled water-treated DPN group. ‡p < 0.05 vs. JMT-treated DPN group. One-way ANOVA followed by Tukey’s multiple comparison test.

Axonal damage was assessed by determining the percentage of APP and beta Tubulin 3 (βIII-tubulin) co-localized axons. APP+ axonal spheroids were clearly visible in longitudinal sections of sciatic nerves from distilled water-treated DPN rats, but not in normal control rats (Figure 4A, 4B, p < 0.01). The percentage of APP+ axonal spheroids was reduced in JMT-treated DPN rats compared to those treated with distilled water (Figure 4A, 4B, p < 0.05).

Figure 4. JMT inhibited APP expression and promoted serum NRG1 level in DPN rats. (A) Representative images of longitudinal sections of sciatic nerves with immunolabeling for APP (green), βIII-tubulin (red), and DAPI (blue, marking nuclei) at a magnification of 400×; scale bars, 20 μm. (B) Percentage of APP+ axonal area with βIII-tubulin colocalization. (C) Panels of serum NRG1 level in different groups. Means ± SD; n=3-8/group. ****p < 0.0001, **p < 0.01 vs. normal control group; ††p < 0.01, †p < 0.05 vs. distilled water-treated DPN group. One-way ANOVA followed by Tukey’s multiple comparison test.

NRG1, a key promoter of neuronal survival, Schwann cell proliferation [30], and myelination [31], is crucial for peripheral nerve growth and development [32]. ELISA results showed that NRG1 level decreased in the serum of distilled water-treated DPN rats (Figure 4C, p < 0.0001), and JMT treatment reversed this effect (Figure 4C, p< 0.01).

Together, these data suggest that JMT exerted a neuroprotective effect against paranodal and axonal pathology with increased serum NRG1 level in DPN rats.

JMT modulated microbiota composition in DPN rats

In order to further explore the mechanisms of JMT’s neuroprotective effects, fecal 16S rRNA gene sequencing and correlation analysis were used to identify differences in gut microbiota composition among normal control rats, distilled water-treated DPN rats, and JMT-treated DPN rats.

As shown in Figure 5, the rarefaction curves of the alpha diversity indexes achieved stability which means the sequencing sample size for gut microbiota analysis is sufficient. Besides, the goods coverage index is very close to 1, which means the sequencing depth has basically covered all the species in the sample.

Figure 5. Rarefaction curves of the alpha diversity indexes in different groups. Rarefaction curves of (A) the Chao1 index, (B) observed species index, (C) Shannon index, (D) Simpson index, (E) PD whole tree index, (F) goods coverage index of fecal samples from different groups. All of the alpha diversity indexes achieved stability in different groups, which means the sequencing sample size for gut microbiota analysis is sufficient and the sequencing depth is enough.

The observed species diversity index (Kruskal-Wallis, p=0.00092, Figure 6A), Shannon diversity index (Kruskal-Wallis, p = 0.00016, Figure 6B) and Simpson diversity index (Kruskal-Wallis, p = 0.00016, Figure 6C) analyses revealed the richness of bacterial colonies and community diversity was reduced in the fecal samples of distilled water-treated DPN rats, compared to normal control rats. However, no significant differences in these alpha diversity indexes were observed between distilled water-treated and JMT-treated DPN rats (Kruskal-Wallis, p > 0.05, Figure 6). Both unweighted (Adonis p=0.001, R²=0.287) and weighted (Adonis p=0.001, R²=0.583) UniFrac-based principal coordinates analysis (PCoA) revealed different microbiota composition clusters in normal control rats, distilled water-treated DPN rats, and JMT-treated DPN rats (Figure 6D, 6E), indicating significantly different beta diversities. Together, these alpha and beta diversity results suggest that JMT might regulate microbiota composition by increasing species diversity among taxa rather than within individual taxa.

Figure 6. JMT modulated microbiota composition in DPN rats. Alpha diversity was evaluated by examining (A) the observed species diversity index, (B) Shannon index, and (C) Simpson index of fecal samples from different groups. Each box plot represents the median, interquartile range, minimum and maximum values. Principal coordinate analysis (PCoA) on (D) unweighted and (E) weighted UniFrac distances among different groups. Boxplots show the distribution of samples along the given axis, representing the median and interquartile range. Ellipses represent a 95% confidence interval around the cluster centroid. Clustering significance by body site was determined by Adonis (p < 0.001).

Linear discriminant analysis coupled with effect size (LEfSe) analysis was performed to identify the specific bacterial taxa differentially represented in normal control rats vs. distilled water-treated DPN rats (Figure 7A) and in distilled water-treated vs. JMT-treated DPN rats (Figure 7B). Compared with normal control rats, distilled water-treated DPN rats were enriched in Bacteroidetes at the phylum level, Bacteroidia at the class level, Bacteroidales at the order level, Porphyromonadaceae, Prevotellaceae, and Oxalobacteraceae at the family level, and Klebsiella, Coprococcus, Prevotella, and Oxalobacter at the genus level, which might be related to the pathogenesis of DPN (Figure 7A). However, we found that JMT treatment could counteract DPN-induced gut dysbiosis by enriching nine species which were also dominant in normal control rats (Figure 7). We therefore speculated that JMT ameliorated gut dysbiosis in distilled water-treated DPN rats by increasing the abundance of Actinobacteria and Proteobacteria at the phylum level, Betaproteobacteria, Actinobacteria, and Epsilonproteobacteria at the class level, Burkholderiales and Campylobacterales at the order level, Helicobacteraceae at the family level, and Helicobacter at the genus level (Figure 7).

Figure 7. Bacterial taxa differences among different groups. Linear discriminant analysis coupled with effect size (LEfSe) analysis was performed to identify the bacterial taxa differentially represented in (A) normal control group vs. distilled water-treated DPN group rats and (B) distilled water-treated DPN group vs. JMT-treated DPN group rats at different taxonomy levels. LDA: linear discriminant analysis, n=8/group.

Association between gut microbiota abundance, DPN phenotypes, and serum NRG1 level

In order to verify the relationship between DPN pathogenesis and gut microbiota, we examined the Spearman’s correlations between alterations in microbiota composition and DPN phenotypes, such as body weight, non-fasting blood glucose level, mechanical withdraw threshold, intraepidermal nerve fiber density, and serum NRG1 level.

As shown in Figure 8, all of the ten species that were enriched in distilled water-treated DPN rats were positively correlated with non-fasting blood glucose level (r > 0.45, p < 0.05), and negatively correlated with body weight (r < -0.45, p < 0.05). Besides, nine of them were also negatively correlated with serum NRG1 level (r < -0.40, p < 0.05); f_Prevotellaceae was not correlated with serum NRG1 level (r > -0.40, p > 0.05). Among the nine species, f_Porphyromonadaceae and g_Prevotella were also negatively correlated with mechanical withdraw threshold (r < -0.40, p < 0.05) and intraepidermal nerve fiber density (r < -0.40, p < 0.05), indicating that these two species might also promote the pathogenesis of DPN. R- and p-values for these correlations are shown in Supplementary Tables 4 and 5. In contrast, the nine species enriched in both JMT-treated DPN rats and normal control rats were all positively correlated with serum NRG1 level (r > 0.40, p < 0.05). In addition, seven of those nine species were positively correlated with mechanical withdraw threshold (r > 0.50, p < 0.05) and intraepidermal nerve fiber density (r > 0.45, p < 0.05); c_Betaproteobacteria and o_Burkholderiales were not correlated with those measures (r <0.35, p > 0.05). Interestingly, among the seven species, p_Actinobacteria, p_Proteobacteria, and c_Actinobacteria were also positively correlated with body weight (r > 0.50, p < 0.01) and negatively correlated with non-fasting blood glucose level (r < -0.55, p < 0.01). These three species might therefore have contributed to JMT induced improvements in DPN phenotypes and symptoms of hyperglycemia and weight loss associated with increased serum NRG1 level (Figure 8, Supplementary Tables 4, 5).

Figure 8. Heatmap of correlations between gut microbiota abundance and DPN phenotypes and serum NRG1 level. The intensity of the color represents the r-value of Spearman’s correlations (negative score, blue; positive score, red) between the relative abundance of the species and DPN phenotypes and serum NRG1 level, n=8/group, +p < 0.05, *p < 0.01.

Drug preparations

JMT is composed of the following 12 kinds of crude drugs: the seeds of Cuscuta chinensis Lam., the seeds of Ligustrum lucidum Ait., the herb of Eclipta prostrata L., the herb of Prunella vulgaris L., the seeds of Litchi chinensis Sonn., Buthus martensii K., the tender stem of Cinnamomum cassia Presl., the rhizoma of Corydalis yanhusuo W. T. Wang, the seeds of Prunus persica L., the seeds of Cassia obtusifolia L, the radix and rhizoma of Asarum heterotropiodes F., and Hirudo nipponica W. at a fixed ratio of 10:10:10:10:30:3:10:10:10:30:3:3. These drug materials were purchased from Beijing Tong Ren Tang Pharm Co., Ltd. (Beijing, China) and authenticated according to the Chinese Flora (http://frps.iplant.cn/frps/Uncaria) and Chinese Pharmacopoeia (Edition 2015, Volume I). Voucher specimens (No. jmt15A - jmt15L) were submitted to the Department of TCM, PUMCH, Beijing, China (Supplementary Table 1) [14]. The chemical profile of JMT was determined by UPLC/Q-TOF-MS analysis using the same formulation batch before use in these experiments; a total of 72 compounds were identified [14] (Supplementary Figure 1, Supplementary Table 2).

Animals

All experiments were performed in accordance with the National Institutes of Health (NIH) “Guide for the Care and Use of Laboratory Animals, 8^th Edition” and were approved by the Animal Ethics Committees of PUMCH [certificate no. XHDW-2018-009]. Specific Pathogen Free (SPF) Sprague Dawley rats (6-weeks old, 160-200 g) were purchased from Vital River (Beijing, China). Upon arrival at the vivarium, rats were quarantined and monitored for one week. Rats were socially housed in SPF clean level conditions using standard rat cages and conventional bedding. Rats were given access to food and water ad libitum. All animal rooms were climate controlled (22–26°C, 40–70% humidity) with a 12-h light/dark cycle [54]. Diabetes was induced by administering freshly dissolved STZ (55 mg/kg in 0.1 mol/L citrate buffer, pH 4.5, i.p.) following an overnight fast. Hyperglycemia was verified 3 days post-STZ (ACCU-CHEK Active; Roche, Basel, Switzerland); rats with non-fasting blood glucose levels ≥ 16.7 mmol/L were classified as diabetic. Diabetic rats were randomly assigned to one of three groups (distilled water-treated DPN rats, JMT-treated DPN rats, or NTP-treated DPN rats; all n=8); age-matched healthy rats were assigned to the normal control group. Rats received 13.9 g/kg/d of crude drug of JMT, 1.6 NU/kg/d of NTP, or 10 mL/kg/d of distilled water daily. JMT and NTP dosages were determined by adjusting the clinical dosage for human adults by a factor equal to the human to rat body surface area ratio. Animals were monitored daily and maintained for 12 weeks post-STZ.

Mechanical withdraw threshold

MWT was assessed 12 weeks post-STZ using the Von Frey Pain Measurement Instrument (cat. no. 2391; IITC Life Science, Woodland Hills, CA, USA). Rats were individually placed in a clear plastic cage containing mesh (1 cm² perforations). After a 15-min acclimation period, the middle of the plantar of each rat was vertically stimulated with the electronic Von Frey probe, making it appear slightly S-shaped, and the paw withdrawal response was observed. A quick flinching reaction immediately after stimulation was considered a positive reaction, and pressure values (grams) were recorded. A paw withdrawal reaction caused by physical activity was not reported as positive. Test was repeated three times at the interval of 15 min and mean value was finally recorded [55].

Blood collection and tissue processing

Blood samples were collected from the abdominal aorta and the serum was stored at -80°C. The bilateral sciatic nerves and plantar skin were cut with sharp scissors and immediately rinsed with ice-cold phosphate buffered saline (PBS) immediately. The sciatic nerve was isolated and cut into two segments: one segment was fixed in 4% paraformaldehyde (PFA) with 2.5% glutaraldehyde in 0.1 M PBS, pH 7.4 at 4°C overnight for ultrastructure observation; the other segment of sciatic nerve and plantar skin were fixed in 4% PFA for pathological and immunohistochemical staining. Rats were sacrificed by cervical dislocation after tissue processing.

Enzyme-linked immunosorbent assay (ELISA)

Serum NRG1 level was measured and calculated using a commercially available kit (Cloud-Clone, Houston, TX, USA) according to the manufacturer’s instructions. Standard or sample, detection reagent, substrate solution and stop solution were sequentially added to wells and incubated with repeated washing as appropriate. Optical density at 450 nm was immediately measured with a plate reader, and sample values were then calculated from the standard curve.

Electron microscopy and morphometric analysis

The 4% PFA with 2.5% glutaraldehyde fixed sciatic nerve tissues were sent to the Electron Microscopy Center in the Centralab Institute of Basic Medical Sciences at the Chinese Academy of Medical Sciences for ultrastructure observation. The nerves were rinsed in PBS, postfixed in 1% osmium tetroxide, embedded in epon, and dehydrated with ethanol and acetone. Ultrathin sections (70 nm) were stained with uranyl acetate and lead citrate and imaged in a transmission electron microscope (TEM-1400, JEOL, Tokyo, Japan) at 8000×, 20000×, and 80000× magnifications. Morphometric parameters such as number of myelinated axons per 5000 μm², percentage of abnormal fibers (fibers with irregular shapes, infoldings, or compacted myelin), and g ratio values (the ratio of inner axon diameter to outer diameter of the myelin sheath, which was used to assess axonal myelination) were analyzed by EM imaging software (RADIUS, EMSIS, Münster, Germany) [56].

Immunohistochemistry

Sciatic nerve tissues fixed in 4% PFA were embedded in paraffin, and cut into 4 μm sections. Sections were stained with hematoxylin and eosin and imaged using an ultra-compact image capture device (Aperio CS2, Leica, Wetzlar, Germany) at 400× magnification. The remaining sciatic nerve sections were treated as follows: regular dewaxing, antigens repair with hot citric acid buffer (pH 6.0), blocking with 10% goat serum. The sections were then incubated overnight with the following primary antibodies at 4°C: Caspr, APP, and βIII-tubulin, followed by the appropriate fluorophore-conjugated secondary antibodies for 40 min at room temperature. Detailed primary and secondary antibody information is shown in Supplementary Table 3. The sections were then washed in PBS and coverslipped in glycerin mounting medium with DAPI. Images were captured using a Nikon A1R confocal system (A1R, Nikon, Tokyo, Japan) at 400× magnification. Quantitative analyses were carried out using Nikon NIS-Element Analyzer.

Intraepidermal nerve fiber density

Fixed plantar skin tissues were washed in PBS containing increasing amounts of sucrose. After washing, the samples were snap-frozen in optimum cutting temperature compound and stored at -80°C. Three longitudinal 50-μm-thick plantar skin sections were blocked with 3% goat serum containing 0.5% porcine gelatin and 0.5% Triton X-100 in Super-Block blocking buffer (Thermo Scientific, Rockford, IL) at room temperature for 2 h. The sections were then incubated overnight with PGP 9.5 at 4°C and then incubated with Alexa Fluor 488 goat anti-rabbit secondary antibody at room temperature for 1 h. Detailed antibody information is shown in Supplementary Table 3. Sections were then coverslipped with glycerin mounting medium. Representative images of intraepidermal nerve fibers were obtained using a Nikon A1R confocal system at 200× magnification. Numbers of intraepidermal nerve fiber profiles and epidermis lengths were assessed using Nikon NIS-Element Analyzer. IENFD was calculated as the number of nerve fiber profiles per millimeter of epidermis [57].

DNA extraction and 16S rRNA gene sequencing

Fecal samples were harvested after an overnight fast at week 12 and then immediately frozen in liquid nitrogen and stored at -80°C. Genomic DNA was extracted from each fecal sample using the E.Z.N.A.^® Stool DNA Kit (OMEGA, Norcross, GA, USA) combined with bead beating according to the manufacturer’s instructions. The 16S rRNA gene sequencing procedure was performed by the Realbio Genomics Institute (Shanghai, China). Extracted genomic DNA was used as the template to amplify the V3-V4 regions of 16S rRNA genes. Amplicon libraries were quantified using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, US) and then sequenced on the Illumina HiSeq platform (Illumina, San Diego, US) with paired-end reads of 250 bp. After discarding singleton reads and removing chimeras, tags were clustered into operational taxonomic units using USEARCH (v7.0.1090) at 97% similarity. A representative sequence of each operational taxonomic unit was then subjected to the taxonomy-based analysis using the RDP database. Heatmap was created using R. Cluster analysis. Alpha diversity and beta diversity were analyzed using QIIME. LEfSe was performed to identify bacterial taxa differentially represented among the groups at different taxonomy levels [58].

Statistical analysis

GraphPad software (San Diego, CA, USA) was used for statistical analysis and graph generation. Data are expressed as mean ± standard deviation unless otherwise indicated. Normally distributed data were analyzed using one-way ANOVA with Tukey’s test for multi-group independent samples. Non-normally distributed data were analyzed using the Kruskal-Wallis test followed by Dunn’s multiple comparisons test for multi-group independent samples. Statistical significance was defined by p < 0.05.