Along with the establishment of more advanced molecular sciences, brand new approaches to managing diabetes mellitus (DM) are anticipated worldwide, especially in overcoming long-term complications. Kidney involvement in DM leads to one of the most common complications. Induced by many factors, inflammation is now known as one of the prime pathogeneses. Previous studies have shown that levels of inflammatory markers can predict the occurrence of DM and its microvascular complications, such as diabetic kidney disease (DKD). ¹ The inflammation pathways vary from oxidative stress, NF-κβ, janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, and inflammatory cytokines. ¹ Overexpression of these markers can lead to vascular dysfunction. NF-κβ, an apoptotic gene, will induce endothelial cell calcification and damage. ² In addition, one of the pro-inflammatory cytokines, TNF-α, is produced by mesangial, endothelial, glomerular, dendritic, and tubular cells in the kidney and majorly induces cytotoxicity, apoptosis, and necrosis. It was explained in a previous study that patients with diabetes mellitus had three to four times higher TNF-α levels compared to healthy adults. ³

In addition to the inflammation control, improving insulin resistance is another main focus in preventing complications in the kidneys due to a prolonged hyperglycemic state. This is supported by the statement that insulin resistance is associated with microalbuminuria. One of the causes of albuminuria is the disruption of podocytes. Podocytes are insulin-sensitive and express glucose transporters, namely GLUT1, GLUT2, GLUT3, GLUT4, and GLUT8. ⁴ GLUTs that are widely discussed are GLUT1 and GLUT4. ^{4 , 5} GLUT4 is the main insulin-induced glucose transporter, whereas GLUT1 acts under basal conditions. Meanwhile, a less-discussed transporter, GLUT2, was also previously known to aid glucose uptake in cultured mouse podocytes. ⁶ Understanding the insulin-signalling process and glucose uptake in podocytes through these GLUTs, especially the far less studied ones, can address new perspectives for novel drug development. ⁶

DLBS3233, an Indonesian herbal product, which is a combined bioactive fraction of Cinnamomum burmanii and Lagerstroaemia speciosa, has demonstrated benefits in glucose control and upregulation of insulin signal transduction. ^{7 – 12} Studies on Swiss Albino 3T3 pre-adipocytes mice showed that DLBS3233 works by increasing the expression of PI3-kinase, Akt, GLUT-4, peroxisome proliferators-activator receptor (PPAR)-γ, and PPAR-δ, and decreased the expression of the resistin gene at the mRNA level. ¹² Previous in vitro studies also reported that DLBS3233 increased GLUT-4 expression leading to increased glucose uptake in insulin-resistant 3T3-Swiss Albino adipocytes. ¹³ Administration of DLBS3233 showed a decrease in HbA1c, fasting blood sugar and blood sugar one hour after fasting, as well as improved insulin resistance, adiponectin levels, low-density lipid (LDL), total cholesterol, and triglycerides. ¹² Following this discovery, further research is required to determine other beneficial effects of this agent, especially in preventing long-term complications in DM. In the present study, we observed in vitro effect of DLBS3233 in diminishing inflammatory markers, specifically TNF-α and NF-κβ, as well as in increasing glucose transporters, namely GLUT1 and GLUT2.

Quantitative IHC comparison between intervention groups is presented in Table 1.

Expression of GLUT1, GLUT2, TNF-α and NF-κβ in IHC can been seen in Figures 1– 4. Figures 1 and 2 show enhanced expression of pancreas GLUT1 and GLUT 2 in experiment groups 3-6, while Figures 3 and 4 display diminished expression of renal TNF-α and NF-κβ.

The IHC analysis of GLUT1 and GLUT2 showed that group 2 experienced a decrease in expression compared to group 1, while GLUT 1 and GLUT2 increased in the treatment group (groups 3, 4, 5 and 6) compared to group 2 (p<0.05) (see Figure 5). Among six groups of intervention, group 2 showed a higher expression of TNF-α and NF-κβ compared to control (p<0.05). Moreover, TNF-αand NF-κβ significantly decreased in the treatment groups (groups 3, 4, 5, and 6) compared to group 2 (p<0.05) (see Figure 6).

DLBS3233 is an Indonesian herbal product whose antidiabetic effect has been explored over the last 10 years. Preclinical studies in Switzerland showed that, at the mRNA level, DLBS3233 increased the expression of the PI3-kinase gene, Akt, GLUT-4, PPAR-γ and PPARδ, and decreased the expression of the resistin gene. Additionally, this substance was discovered to boost GLUT4 synthesis and activate adiponectin transcription at the protein level, consequently enhancing glucose uptake. ^{14 , 15} However, little research has been done on how DLBS3233 affects the kidneys.

DM is a metabolic disease with a systemic effect on the kidneys, through an inflammatory process that damages the microvasculature, which in turn induces podocyte damage, characterized by proteinuria. ³ TNF-alpha increases in diabetic patients, triggering inflammation which later damages podocytes. The inflammation also results in an endothelial dysfunction through NF-kb, causing endothelial cell calcification. ^{2 , 3} This endothelial cell calcification might lead to the surge of oxidative stress and fibrosis in the process of diabetic nephropathy. ¹⁶ The decrease in TNF-alpha and NF-kb in diabetes certainly prevents microvascular damage and fibrosis in the kidneys.

GLUT are glucose transporters that help insulin in the uptake of glucose to cells. GLUT1 acts on basal membrane, whereas GLUT2 acts on glucose uptake in podocytes. In diabetic patients, the decrease in each transporter indicates the process of insulin resistance. ^{4 – 6} DLBS3233 has been reported to increase GLUT4 synthesis which improves the glycemic profile of diabetic patients. ¹² Our study showed that in the diabetic group, GLUT1 and GLUT2 were significantly lowered compared to the normal group. Administration of DLBS3233, showed an increase in the expression of GLUT 1 and GLUT2 through IHC examination of cells in the pancreas.

Besides, we also examined IHC for the expression of TNF-α and NF-κβ in the kidneys. In the group of diabetic rats, TNF-α and NF-κβ increased; on the other hand, there was a significant decline of both inflammatory markers in the treatment group. DLBS3233, apart from improving sugar uptake, has also been proven to improve inflammation in the kidneys.

Our experiment was a preliminary study and was still limited to the expression of GLUT1 and GLUT2 in the pancreas, with TNF- and NF-κβ expression in the kidney. Further research of other effects of DLBS3233 in the kidneys is underway.

DLBS3233 administration stimulated the decline of inflammatory markers (TNF-alfa and NF-kb) in the kidneys as well as enhancing the glucose uptake through increasing pancreatic GLUT1 and GLUT2 expression.