6-Gingerol, Metformin, and their combination attenuated renal injury and improved renal functions in diabetic rats

Serum creatinine and BUN concentrations, urinary protein excretion level, creatinine clearance, and proteinuria/creatininuria ratio were measured to assess the renoprotective effects of 6-gingerol, metformin, and their combination. As shown in Fig. 2A, B, and C, diabetic rats exhibited a 2.8-fold, 2-fold, and 5.8-fold elevation of serum creatinine, BUN, and urinary protein excretion levels, respectively, corresponding to control rats (p < 0.0001). Administration of 6-gingerol, metformin, and their combination to diabetic rats brought about a significantly reduced serum creatinine concentration by 37.7%, 40.6%, and 60.8%, respectively, and BUN by 38.7%, 42.2%, and 49.8%, respectively, while urinary protein excretion levels were reduced by 51.4%, 62.6%, and 77.1%, respectively. Besides, serum creatinine and BUN concentrations, as well as urinary protein excretion levels, were significantly reduced upon treatment of diabetic rats with 6-gingerol + metformin combination as compared to those treated with either 6-gingerol (p < 0.001), (p < 0.001), (p < 0.0001) or metformin (p < 0.01), (p < 0.05) and (p < 0.01), respectively. Compared to the control group, 6-gingerol and metformin groups showed a significant increase in serum creatinine (p < 0.0001) and (p < 0.001), respectively, and BUN (p < 0.0001) concentrations and urinary protein excretion level (p < 0.0001), while no significant difference was found in 6-gingerol + metformin group with respect to control group.

Fig. 2

GR, MET, and their combination attenuated renal injury and improved renal functions in diabetic rats

Renal functions assessment by measuring serum levels of A: serum creatinine, B: blood urea nitrogen (BUN); C: urinary protein excretion level; D: creatinine clearance; and E: proteinuria/creatininuria ratio. DN: diabetic nephropathy; GR: 6-gingerol; MET: metformin. Data are represented as Mean \( \pm \) SEM (*p\( <\) 0.05, **p\( <\) 0.01, ***p\( < \)0.001, ****p\( <\) 0.0001, ns: non-significant)

On the other hand, Fig. 2D showed that diabetic rats exhibited a 2.5-fold reduction in creatinine clearance relative to the control group (p < 0.0001). Creatinine clearance was significantly elevated in 6-gingerol (p < 0.01), metformin (p < 0.001), and 6-gingerol + metformin combination (p < 0.0001) groups relative to the DN group. Besides, creatinine clearance manifested a significant elevation in diabetic rats receiving the 6-gingerol + metformin combination when compared to those treated with 6-gingerol (p < 0.001) or metformin (p < 0.05) alone. Compared to the control group, 6-gingerol and metformin groups showed a significant decrease in creatinine clearance (p < 0.0001) and (p < 0.001), respectively, while no significant difference was found in 6-gingerol + metformin group with respect to control group.

Furthermore, Fig. 2E demonstrated a 3.9-fold elevation in proteinuria/creatininuria ratio in diabetic rats with respect to control group (p < 0.0001). Proteinuria/creatininuria ratio was markedly reduced in 6-gingerol, metformin, and 6-gingerol + metformin combination groups relative to the DN group (p < 0.0001). In addition, proteinuria/creatininuria ratio exhibited a significant reduction in diabetic rats receiving the 6-gingerol + metformin combination when compared to those treated with 6-gingerol (p < 0.001) or metformin (p < 0.05) alone. Also, proteinuria/creatininuria ratio was significantly elevated in 6-gingerol and metformin groups with respect to control group (p < 0.0001) whereas a non-significant difference was observed in 6-gingerol + metformin group relative to the control group. Interestingly, renal functions were markedly improved in rats treated with the 6-gingerol + metformin combination, as indicated by the non-significant variation in serum creatinine and BUN concentrations, urinary protein excretion level, creatinine clearance, and proteinuria/creatinuria ratio with respect to control rats.

The renoprotective effects of the 6-gingerol, metformin, and 6-gingerol + metformin combination were further examined in kidney tissue segments stained with H&E (Fig. 3A and B). Microscopic pictures from the control group showed normal kidney architecture in both the cortex and medulla, with normal glomerular and tubular structures. Severe histopathological lesions were observed in the DN group in both cortex and medulla, including diffuse tubular hydropic degeneration, tubular necrosis, hyaline casts, congested glomeruli, and congested inter-tubular blood vessels. 6-Gingerol group showed moderately decreased histopathological lesions as compared to the DN group (p < 0.05) and exhibited moderate tubular hydropic degeneration, hyaline casts, and congested inter-tubular blood vessels. Metformin group showed moderately decreased histopathological lesions compared to the DN group (p < 0.05) including moderate tubular hydropic degeneration, congested glomeruli, and mildly congested inter-tubular blood vessels. The 6-gingerol + metformin group showed a significantly improved histopathology relative to DN group (p < 0.001) and displayed mild tubular hydropic degeneration and mildly congested inter-tubular blood vessels which seemed non-significantly different from the control group.

Fig. 3

Histopathological examination of H&E-stained renal sections showing renoprotective effects of GR, MET, and their combination

A: Microscopic images of hematoxylin and eosin (H&E)-stained renal sections showing normal cortex and medulla in the control group, severe pathological changes in the cortex and medulla in DN group, moderately decreased pathological changes in the cortex and medulla in GR group and MET group, and markedly improved histological picture with mild pathological changes in the cortex and medulla in GR + MET group. Black arrows: diffuse tubular hydropic degeneration, dashed arrows: tubular necrosis, black arrowheads: cast formation, circular arrows: congested glomeruli, elbow arrows: congested inter-tubular blood vessels. X: 400, scale bar = 50 micrometer

B: Renal histopathological changes were assessed semi-quantitatively and given scores from 0 to 3, where 0 is normal, 1 is mild, 2 is moderate, and 3 is severe. DN: diabetic nephropathy, GR: 6-gingerol, MET: metformin. Data are represented as median and range (*p\( <\) 0.05, ***p\( < \)0.001, ****p\( <\) 0.0001, ns: non-significant)

6-Gingerol, Metformin, and their combination down-regulated TLR4/TRAF6/NLRP3 inflammasome pathway gene expression in DN

As shown in Fig. 4A, B, C, D, and E, the DN group revealed a significantly increased mRNA expression of each of TLR4 (3.6-fold), TRAF6 (4.3-fold), NF-κB (p65) (4.2-fold), NLRP3 (3.4 fold), and caspase-1 (4.8 fold) relative to the control group (p < 0.0001). Diabetic rats treated with 6-gingerol, metformin, and 6-gingerol + metformin combination showed significantly lower mRNA expressions of TLR4 by 52.5%, 55%, and 68%, respectively; TRAF6 by 39.5%, 51.1%, and 74.2%; NF-κB (p65) by 46.6%, 50.1%, and 70.5%; NLRP3 by 47.2%, 53.2%, and 67.9%; and caspase-1 by 36.1%, 47%, and 71.2%, respectively.

Fig. 4

GR, MET, and their combination down-regulated TLR4/TRAF6/NLRP3 inflammasome pathway gene expression in DN

Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure mRNA relative expression of TLR4 (A), TRAF6 (B), NF-κB (p65) (C), NLRP3 (D), and caspase-1 (E) in renal tissue. DN: diabetic nephropathy; GR: 6-gingerol; MET: metformin; TLR4: Toll-like receptor 4; TRAF6: Tumor necrosis factor receptor-associated Factor 6; NF-κB (p65): Nuclear factor-kappa B (p65); NLRP3: NOD-like receptor family pyrin domain-containing 3. Data are represented as Mean \( \pm \) SEM (***p\( < \)0.001, ****p\( <\) 0.0001, ns: non-significant)

In addition, the 6-gingerol + metformin group displayed significantly lower TLR4, TRAF6, and NF-κB (p65) mRNA expressions relative to 6-gingerol (p < 0.0001) or metformin (p < 0.001) group and a significant decrease in NLRP3 and caspase-1 mRNA expressions as compared to 6-gingerol or metformin group (p < 0.0001). 6-Gingerol and metformin groups showed a significant increase in TLR4, TRAF6, NF-κB (p65), NLRP3, and caspase-1 mRNA expressions relative to control group (p < 0.0001) while no significant variation was seen between 6-gingerol + metformin group and control group.

6-Gingerol, Metformin, and their combination suppressed inflammation and pyroptosis in diabetic kidneys

In addition to gene expression, TLR4, TRAF6, NF-κB (p65), NLRP3, and caspase-1 protein levels were further determined in renal tissue. As shown in Fig. 5A, B, C, and D, diabetic rats revealed a 4-fold elevation in TLR4, a 4.5-fold elevation in TRAF6, a 3.4-fold elevation in NLRP3, and a 4.6-fold elevation in caspase-1 levels relative to the control group (p < 0.0001). Administering 6-gingerol, metformin, or 6-gingerol + metformin combination to diabetic rats brought about significantly reduced levels of TLR4 by 38%, 47.3%, and 70.5%, respectively; TRAF6 by 32.3%, 37.6%, and 58.1%; NLRP3 by 40.4%, 36.6%, and 57.9%; and caspase-1 by 45.2%, 50.5%, and 70.5%, respectively. Furthermore, the 6-gingerol + metformin group revealed significantly reduced levels of TLR4 (p < 0.001), (p < 0.05); TRAF6 (p < 0.001), (p < 0.01); NLRP3 (p < 0.05), (p < 0.01); and caspase-1 (p < 0.0001) as compared to either 6-gingerol or metformin groups, respectively. In comparison with the control group, 6-gingerol and metformin groups showed significantly elevated levels of TLR4 (p < 0.0001), (p < 0.01); TRAF6 (p < 0.0001); NLRP3 (p < 0.0001); and caspase-1 (p < 0.0001). A non-significant difference was found between 6-gingerol + metformin group and control group regarding TLR4, NLRP3, and caspase-1 levels whereas TRAF6 level was significantly higher in 6-gingerol + metformin group compared to control group (p < 0.05).

Fig. 5

GR, MET, and their combination suppressed inflammation and pyroptosis in diabetic kidneys

Renal tissue protein levels of TLR4 (A), TRAF6 (B), NLRP3 (C), caspase-1 (D), TNF-α (E), and IL-1β (F). DN: diabetic nephropathy; GR: 6-gingerol; MET: metformin; TLR4: Toll-like receptor 4; TRAF6: Tumor necrosis factor receptor-associated Factor 6; NLRP3: NOD-like receptor family pyrin domain-containing 3; TNF-α: tumor necrosis factor-alpha; IL-1β: interleukin-1 beta. Data are represented as Mean \( \pm \) SEM (*p\( <\) 0.05, **p\( <\) 0.01, ***p\( < \)0.001, ****p\( <\) 0.0001, ns: non-significant)

6-Gingerol, metformin, and their combination decreased renal inflammatory cytokines levels and diminished the inflammatory response in DN

Since inflammatory cytokines are crucial for the onset and development of DN, protein levels of TNF-α and IL-1β were measured in kidney tissues. Figure 5E and F demonstrated a significant elevation in renal TNF-α and IL-1β levels in the DN group relative to the control group (p < 0.0001). A significant reduction of TNF-α level was observed in 6-gingerol, metformin, and 6-gingerol + metformin combination groups by 40.5%, 43.4%, and 52.6%, respectively, while IL-1β level was significantly decreased by 37.2%, 44.7%, and 63.1%, respectively. The 6-gingerol + metformin combination group exhibited a significantly reduced TNF-α level with regards to 6-gingerol (p < 0.001) and metformin (p < 0.01) groups. Also, IL-1β expressed a markedly reduced level in the 6-gingerol + metformin group in comparison with 6-gingerol and metformin groups (p < 0.0001). As compared to control group, 6-gingerol and metformin groups manifested significantly elevated TNF-α (p < 0.0001), (p < 0.001) and IL-1β (p < 0.0001) levels, respectively, whereas 6-gingerol + metformin group showed no significant difference as compared to control group.

6-Gingerol, Metformin, and their combination suppressed NF-κB (p65) protein expression in kidney tissue

Protein expression of NF-κB (p65) in kidney tissue was examined by immunohistochemistry (Fig. 6A and B). Microscopic images of immunostained renal sections against NF-κB (p65) showed no brown staining in either the cortex or medulla in the control group. DN group showed excess brown tubular staining in both the cortex and medulla which implied an elevated expression of NF-κB (p65), as well as excess brown nuclear staining in tubules, which revealed the elevated nuclear expression of activated NF-κB (p65). NF-κB (p65) expression was semi-quantified by measuring NF-κB (p65) positive area percentage, which was significantly elevated in the DN group relative to the control group (p < 0.0001). Administration of 6-gingerol, metformin, or 6-gingerol + metformin combination to diabetic rats resulted in a significant reduction in NF-κB (p65) positive area percentage with regards to untreated diabetic rats (p < 0.0001). 6-Gingerol and metformin groups exhibited a significant elevation in NF-κB (p65) positive area percentage in comparison with control group (p < 0.0001). Moreover, the 6-gingerol + metformin group exhibited a significantly decreased NF-κB (p65) positive area percentage when compared with either 6-gingerol or metformin group (p < 0.0001) and a non-significant difference with respect to the control group.

Fig. 6

Immunohistochemical examination of NF-κB (p65) in renal tissue

A: Microscopic images of immunostained renal sections against NF-κB (p65) showing negative staining in the cortex and medulla in the control group, excess brown tubular staining in the cortex and medulla in the DN group, decreased brown tubular staining in the cortex and medulla in GR group and MET group, and much more decreased brown tubular staining in the cortex and medulla in GR + MET group. Black arrows refer to nuclear staining. Immunohistochemistry is counterstained with Mayer’s hematoxylin. X: 400, scale bar = 50 micrometer

B: Immunostaining of NF-κB (p65) was assessed quantitatively through the area of positive expression. DN: diabetic nephropathy, GR: 6-gingerol, MET: metformin, NF-κB (p65): nuclear factor-kappa B (p65). Data are represented as Mean \( \pm \) SEM (****p\( <\) 0.0001, ns: non-significant)

6-Gingerol, Metformin, and their combination up-regulated miRNA-146a and miRNA-223 gene expression in diabetic kidneys

As shown in Fig. 7A and B, a significant down-regulation of miRNA-146a (4.4-fold) and miRNA-223 (2.8-fold) gene expression was observed in the DN group in comparison with the control group (p < 0.0001). When compared to the DN group, miRNA-146a gene expression was significantly higher in the 6-gingerol, metformin, and 6-gingerol + metformin groups (p < 0.0001). Also, miRNA-223 gene expression was significantly higher in 6-gingerol (p < 0.01), metformin (p < 0.0001), and 6-gingerol + metformin (p < 0.0001) groups with respect to the DN group. In addition, the 6-gingerol + metformin group revealed significantly up-regulated miRNA-146a and miRNA-223 gene expressions with respect to either the 6-gingerol or metformin group (p < 0.0001). Moreover, miRNA-146a and miRNA-223 were significantly decreased in 6-gingerol and metformin groups with respect to control group (p < 0.0001), while no significant difference was observed in 6-gingerol + metformin group with respect to control group.

Fig. 7

GR, MET, and their combination up-regulated miRNA-146a and miRNA-223, decreased oxidative stress and lipid peroxidation

Relative expression of miRNA-146a (A) and miRNA-223 (B) was measured using quantitative real-time polymerase chain reaction (qRT-PCR). Oxidative stress was assessed by spectrophotometric measurement of the levels of reduced glutathione (GSH) (C) and malondialdehyde (MDA) (D) in renal tissue and the levels of serum MDA (E) and urinary MDA (F). DN: diabetic nephropathy, GR: 6-gingerol; MET: metformin. Data are represented as Mean \( \pm \) SEM (*p\( <\) 0.05, **p\( <\) 0.01, ***p\( <\) 0.001, ****p\( <\) 0.0001, ns: non-significant)

6-Gingerol, Metformin, and their combination decreased oxidative stress in diabetic kidneys

Our results revealed a markedly improved oxidative status in diabetic rats that received 6-gingerol, metformin, or their combination. As shown in Fig. 7C, a significant reduction in renal GSH level was observed in the DN group relative to the control group (p < 0.0001) reflecting a state of high oxidative stress in diabetic rats. Administration of 6-gingerol, metformin, and 6-gingerol + metformin combination to diabetic rats brought about significantly elevated renal GSH levels by 71.2%, 50.2%, and 93%, respectively. Renal GSH level was markedly elevated in the 6-gingerol group relative to the metformin group (p < 0.05). Both 6-gingerol and metformin groups showed a significant decrease in renal GSH level (p < 0.01) and (p < 0.0001), respectively, compared to the control group. The 6-gingerol + metformin group revealed significantly elevated renal GSH levels when compared with 6-gingerol (p < 0.001) and metformin (p < 0.0001) groups and no significant difference relative to the control group.

Alternatively, Fig. 7D, E and F demonstrated significantly elevated MDA levels in renal tissue, serum and urine, respectively, in the DN group with respect to the control group (p < 0.0001) reflecting a significant increase in lipid peroxidation. Administration of 6-gingerol, metformin, and 6-gingerol + metformin combination to diabetic rats led to significantly reduced renal MDA levels by 30.8%, 21.4%, and 45.5%; serum MDA levels by 27.2%, 22.9%, and 44%; and urine MDA levels by 36.4%, 25.9%, and 66.7%, respectively. Renal MDA level was markedly lower in the 6-gingerol group relative to the metformin group (p < 0.05). The MDA levels were significantly reduced in renal (p < 0.0001), serum (p < 0.01), and urinary (p < 0.0001) samples in 6-gingerol + metformin group with respect to 6-gingerol group. The 6-gingerol + metformin group exhibited significantly lower renal, serum, and urinary MDA levels with respect to metformin group (p < 0.0001). Compared with control group, renal and urinary MDA levels were significantly higher in both 6-gingerol and metformin groups (p < 0.0001) and serum MDA level was significantly higher in 6-gingerol (p < 0.01) and metformin (p < 0.0001) groups. Besides, the 6-gingerol + metformin group displayed non-significantly different renal and urinary MDA levels and significantly higher serum MDA level (p < 0.01) when compared with the control group.

6-Gingerol, Metformin, and their combination suppressed renal fibrosis in diabetic kidneys

As demonstrated by Fig. 8A and B, Masson trichrome-stained renal tissue segments showed no excess collagen deposition in either the cortex or medulla in the control group. DN group showed excess bluish-stained collagen deposition in the renal cortex and medulla, reflecting significant fibrosis in the DN group. 6-Gingerol and metformin groups showed markedly less bluish-stained collagen deposition. The collagen deposition was even more significantly reduced in the 6-gingerol + metformin group. The fibrosis percentage was markedly decreased by 75.8% in the 6-gingerol group and by 79.3% in the metformin group relative to the DN group. 6-Gingerol and metformin groups exhibited a significant elevation in fibrosis percentage in comparison with control group (p < 0.001). In addition, the 6-gingerol + metformin group demonstrated a significantly decreased fibrosis percentage relative to either 6-gingerol (p < 0.001) or metformin (p < 0.01) groups and non-significant differences with respect to the control group.

Fig. 8

Histopathological examination of Masson’s trichrome-stained renal sections and fibrosis percentage assessment in diabetic rats

A: Microscopic images of Masson’s trichrome-stained renal sections showing no collagen deposition in the cortex or medulla in the control group, excess bluish-stained collagen deposition in the cortex and medulla in the DN group, decreased collagen deposition in the cortex and medulla in the GR group and MET group, and much more decreased collagen deposition in the cortex and medulla in GR + MET group. Black arrows refer to collagen deposition. X: 400, scale bar = 50 micrometer

B: Fibrosis percentage was assessed quantitatively through Masson’s-positive area percentage. DN: diabetic nephropathy; GR: 6-gingerol; MET: metformin. Data are represented as Mean \( \pm \) SEM (**p\( <\) 0.01, ***p\( < \)0.001, ****p\( <\) 0.0001, ns: non-significant)

Fibronectin deposition in kidney tissue was stained immunohistochemically to further evaluate renal fibrosis (Fig. 9A and B). Microscopic images of immunostained kidney tissue segments against fibronectin showed no excess brown staining in the cortex and medulla in the control group. Meanwhile, excess brown tubular staining was expressed by the DN group in both the cortex and medulla, reflecting significantly increased fibronectin deposition relative to the control group (p < 0.0001). All treatment groups showed a remarkable reduction in brown tubular staining in both the cortex and medulla. 6-Gingerol and metformin groups demonstrated significantly decreased fibronectin deposition by 71.1% and 84.8%, respectively, in comparison with the DN group. However, 6-gingerol and metformin groups exhibited a significant elevation in fibronectin deposition in comparison with control group (p < 0.0001). The 6-gingerol + metformin group demonstrated significantly decreased fibronectin deposition when compared with 6-gingerol and metformin groups (p < 0.0001) and non-significantly different fibronectin deposition relative to the control group.

Fig. 9

Immunohistochemical examination of fibronectin and gene expression of HIF-1α in renal tissue

A: Microscopic images of immunostained renal sections against fibronectin showing negative staining in the cortex and medulla in the control group, excess brown tubular staining in the cortex and medulla in DN group, decreased brown tubular staining in the cortex and medulla in GR group and MET group, and much more decreased positive brown tubular staining in the cortex and medulla in GR + MET group. Black arrows refer to fibronectin deposition. Immunohistochemistry is counterstained with Mayer’s hematoxylin. X: 400, scale bar = 50 micrometer

B: Immunostaining of fibronectin was assessed quantitatively through the area of positive expression. DN: diabetic nephropathy, GR: 6-gingerol, MET: metformin. Data are represented as Mean \( \pm \) SEM (****p\( <\) 0.0001)

C: Renal hypoxia was assessed by measuring mRNA relative expression of hypoxia-inducible factor-1 alpha (HIF-1α). DN: diabetic nephropathy, GR: 6-gingerol, MET: metformin. Data are represented as Mean \( \pm \) SEM (***p\( < \)0.001, ****p\( <\) 0.0001, ns: non-significant)

Fig. 10

A summary figure of the mechanisms of action of GR, MET, and their combination in HFD/STZ-induced DN in rats

DN: diabetic nephropathy; GR: 6-gingerol; MET: metformin; HFD: high-fat diet; STZ: streptozotocin; TLR4: Toll-like receptor 4; TRAF6: tumor necrosis factor receptor-associated Factor 6; NF-κB (p65): nuclear factor-kappa B (p65); NLRP3: NOD-like receptor family pyrin domain-containing 3; TNF-α: tumor necrosis factor-alpha; IL-1β: interleukin-1 beta; ROS: reactive oxygen species; GSH: reduced glutathione; MDA: malondialdehyde; HIF-1α: hypoxia-inducible factor-1 alpha.

6-Gingerol, Metformin, and their combination diminished renal hypoxia in DN

As shown in Fig. 8C, the DN group showed a 2.9-fold elevation in the mRNA expression of HIF-1α with respect to the control group (p < 0.0001). This elevation was significantly reduced when diabetic rats received 6-gingerol, metformin, or 6-gingerol + metformin combination by 46.4%, 40.4%, and 61%, respectively (p < 0.0001). However, 6-gingerol and metformin groups still exhibited a significant elevation in HIF-1α mRNA expression in comparison with control group (p < 0.0001). A marked reduction in HIF-1α mRNA expression was seen in the 6-gingerol + metformin group relative to 6-gingerol (p < 0.001) or metformin group (p < 0.0001) and a non-significant variation was found with respect to the control group.

6-Gingerol, Metformin, and their combination reduced fasting blood glucose and renal hypertrophy in DN

As shown in Table 2, DN group expressed a 4.2-fold elevated fasting blood glucose level relative to the control group (p < 0.0001). When diabetic rats were administered 6-gingerol, metformin, or 6-gingerol + metformin combination, fasting blood glucose was markedly lowered by 54.9%, 64.3%, and 74.2%, respectively. Noteworthy, fasting blood glucose was significantly lower in the 6-gingerol group relative to the metformin group (p < 0.05). 6-Gingerol + metformin combination group revealed a significantly decreased fasting blood glucose with regards to 6-gingerol (p < 0.0001) or metformin (p < 0.05) groups. Fasting blood glucose was still significantly elevated in 6-gingerol (p < 0.0001) and metformin (p < 0.01) groups relative to control group. Whereas the fasting blood glucose became normalized in rats receiving the 6-gingerol + metformin combination.

Table 2 6-gingerol (GR), metformin (MET), and GR + MET effects on blood glucose, KW/BW index, and lipid profile

DN group showed a 32% increase in kidney weight/body weight (KW/BW) index relative to the control group (p < 0.0001), which reveals marked renal hypertrophy in diabetic rats (Table 2). Administration of 6-gingerol, metformin, and 6-gingerol + metformin combination to diabetic rats has significantly decreased the KW/BW index by 22.4%, 22.1%, and 32.4%, respectively. The 6-gingerol + metformin combination group exhibited a significantly decreased KW/BW index relative to 6-gingerol (p < 0.01) or metformin (p < 0.05) groups. KW/BW index was still significantly elevated in 6-gingerol (p < 0.0001) and metformin (p < 0.001) groups relative to control group whereas it was restored in diabetic rats treated with the 6-gingerol + metformin combination, i.e., no significant difference was found when compared to normal control rats.

6-Gingerol, Metformin, and their combination improved lipid profile in diabetic rats

Our results demonstrated an overall improvement in the lipid profile of diabetic rats receiving 6-gingerol, metformin, or their combination. As shown in Table 2, diabetic rats showed 2.6-fold, 2.1-fold, and 5.1-fold elevation in the levels of serum triglycerides, total cholesterol, and LDL cholesterol, respectively, as well as a 1.7-fold decrease in HDL cholesterol levels in comparison with control rats. 6-Gingerol, metformin, and 6-gingerol + metformin combination have significantly decreased triglycerides, total cholesterol, and LDL cholesterol (p < 0.0001) while significantly increased HDL cholesterol levels (p < 0.01), (p < 0.0001), and (p < 0.0001), respectively, with regards to the DN group. In addition, the 6-gingerol + metformin group revealed significantly decreased triglycerides (p < 0.05), (p < 0.01), and total cholesterol (p < 0.01), (p < 0.05) levels relative to 6-gingerol and metformin groups, respectively. The 6-gingerol + metformin group revealed significantly lower LDL cholesterol levels with regards to the 6-gingerol group (p < 0.01) and non-significantly lower LDL cholesterol levels with regards to the metformin group. No significant difference in HDL cholesterol existed in diabetic rats treated with the 6-gingerol + metformin combination relative to those treated with 6-gingerol or metformin. In comparison with the control group, 6-gingerol and metformin groups showed a significant elevation in serum triglycerides (p < 0.01) and (p < 0.001), total cholesterol (p < 0.001), and LDL cholesterol (p < 0.0001) and (p < 0.001), respectively. Regarding HDL cholesterol, a significant decrease was found in 6-gingerol group (p < 0.01) and a non-significant decrease was observed in metformin group, in comparison with the control group. Furthermore, no significant difference was observed between 6-gingerol + metformin group and control group regarding serum triglycerides, total cholesterol, LDL cholesterol and HDL cholesterol.