Renoprotective Effects Of A Cistanche-Based Bushen Kaixuan Tongluo Formula in Db/db Mice Via Activation Of The CAMP/PKA/CREB Signaling Pathway

3. Results

3.1 Effects of the Cistanche-Based Bushen Kaixuan Tongluo Formula on General Condition of db/db Mice

Mice in the blank group were active and had glossy fur. In contrast, mice in the model group appeared obese, lethargic, and significantly less active. Mice in the treatment groups showed varying degrees of improvement in general condition compared to the model group.

 

 

 

Cistanche-Based Bushen Kaixuan Tongluo Formula on Kidney and Liver Health

 

3.2 Effects of the Formula on Body Weight of db/db Mice

Throughout the experimental period, the body weight of mice in the model group increased significantly compared to the blank group (P<0.01). Compared with the model group, mice in all treatment groups showed a downward trend in body weight, with differences appearing from week 8 onward. The high-dose Cistanche-based formula group showed the most pronounced effect. (See Table 2)

3.3 Effects on Renal Function

Compared to the blank group, serum creatinine (Scr) in the model group showed an upward trend, though not statistically significant, while blood urea nitrogen (BUN), urinary albumin (uALB), and albumin-to-creatinine ratio (ACR) were significantly elevated (P<0.05). Compared with the model group, mice in the formula-treated groups showed a decreasing trend in Scr (not significant), and significantly reduced levels of BUN, uALB, and ACR (P<0.05), with the medium-dose group showing the best results. In the irbesartan group, Scr and BUN showed a downward trend (not significant), while uALB and ACR were significantly reduced (P<0.01). (See Table 3)

3.4 Effects on Liver Function and Lipid Metabolism

Compared with the blank group, ALT, AST, total cholesterol (TC), and triglycerides (TG) were significantly increased in the model group (P<0.05). Compared with the model group, the treatment groups showed a decreasing trend in AST and ALT (not significant), and a significant decrease in TC (P<0.01). TG levels were significantly reduced in the medium-dose formula group (P<0.05), while other formula groups showed a downward trend (not significant). In the irbesartan group, TC showed a decreasing trend (not significant), and TG was significantly reduced (P<0.01). (See Table 4)

3.5 Effects on Glucose Metabolism

Compared with the blank group, fasting blood glucose (FBG), leptin (LEP), glycosylated serum protein (GSP), and insulin (INS) levels were significantly elevated in the model group (P<0.05). Compared with the model group, FBG showed no significant downward trend in any group; LEP levels decreased (not significant) in the formula groups, whereas irbesartan significantly reduced LEP (P<0.05). All treatment groups showed significantly reduced GSP and INS levels (P<0.05), with the high-dose Cistanche-based formula group showing the best effect. (See Table 5)

 

3.6 Effects on Renal Histopathology

HE staining: The blank group showed normal renal architecture with regular glomerular morphology and intact tubular epithelial cells. The model group exhibited irregular glomeruli, moderate mesangial cell proliferation, inflammatory infiltration, and vacuolar degeneration in tubular epithelial cells. These pathological features were alleviated in the low-, medium-, and high-dose formula and irbesartan groups. (See Figure 1)

PAS staining: The blank group showed minimal extracellular matrix (ECM) deposition. The model group had marked ECM accumulation and thickened basement membranes. The treatment groups showed varying degrees of improvement. (See Figure 2)

Masson staining: The blank group revealed normal glomerular structure with minimal collagen deposition. The model group showed extensive blue-stained collagen in glomeruli and renal interstitium. The pathological changes were alleviated in all treatment groups. (See Figure 3)

 

3.7 Effects on p-PKA and p-CREB Expression in Renal Tissue

Compared with the blank group, p-PKA and p-CREB staining in the renal tissues of the model group was significantly reduced (P<0.01). Compared with the model group, the medium-dose formula group had significantly increased p-PKA expression (P<0.01), and p-CREB showed an upward trend (not statistically significant). (See Figure 4, Table 6)

 

3.8 Effects on p-PKA, PKA, p-CREB, CREB, and Bcl-2 Protein Expression

Compared with the blank group, the model group showed significantly decreased expression of p-PKA/PKA, p-CREB/CREB, and Bcl-2 (P<0.05). These levels were significantly elevated in the medium-dose Cistanche-based formula group (P<0.01). (See Figure 5, Table 7)

 

4. Discussion

Although traditional Chinese medicine (TCM) literature does not explicitly describe diabetic kidney disease (DKD), symptoms like foamy urine align with TCM conditions such as "urine turbidity" and "guan ge." Based on decades of clinical experience, Professor Lü Cuiyan developed the Bushen Kaixuan Tongluo Formula, using Cistanche in place of Astragalus as the monarch herb for tonifying Qi and Yang, paired with Aconite to warm the kidney and transform turbidity.

Salvia and Hirudo act as minister herbs to activate blood and unblock collaterals, while Saposhnikovia, Bombyx batryticatus, and cicada slough expel wind and pathogens. Rehmannia and Cornus nourish kidney essence; Achyranthes guides herbs downward; Rosa laevigata, Euryale, Rubus, and Cnidium consolidate the essence; Dioscorea strengthens spleen and kidney. The combination achieves spleen-kidney tonification, turbidity removal, and stasis resolution, showing promising clinical efficacy.

Modern pharmacological studies indicate the formula's components act on multiple targets to improve renal function:

Tanshinone IIA and hirudin inhibit TGF-β/Smad signaling, reducing fibrosis and microcirculatory disorders

Saposhnikovia polysaccharides downregulate NF-κB-mediated inflammation

Iridoid glycosides from Cornus activate the Nrf2/ARE pathway to enhance antioxidant enzymes

Achyranthes sterols modulate PI3K/Akt pathway, inhibiting podocyte apoptosis

Dioscin activates AMPK/mTOR to regulate autophagy and epithelial cell metabolism

The cAMP signaling pathway, central to cellular energy metabolism and gene expression, is vital for renal homeostasis. Adenylate cyclase (AC) generates cAMP, which allosterically activates PKA; activated PKA phosphorylates CREB to form p-CREB, which translocates to the nucleus and initiates transcription of target genes like Bcl-2, promoting anti-apoptotic effects and reducing renal injury.

In DKD, sustained hyperglycemia disrupts cAMP balance by inhibiting AC function and enhancing PDE-mediated degradation, leading to decreased cAMP levels. This impairs PKA/CREB signaling, compromises mitochondrial function, antioxidant defense, and anti-apoptotic gene expression, accelerating glomerular sclerosis and interstitial fibrosis.

Research shows that activating the cAMP/PKA pathway helps maintain podocyte slit diaphragm integrity and reduce proteinuria. Moreover, CREB transcriptionally induces antioxidant enzymes like SOD2, mitigating tubular oxidative stress. Bcl-2 also reduces mitochondrial ROS leakage, protecting podocytes and tubular cells under hyperglycemia.

Restoring normal glucose levels reduces AGE accumulation, alleviating basal membrane thickening and podocyte injury. Improving lipid profiles reduces renal lipid deposition and macrophage infiltration, thus delaying fibrosis progression.

However, research on how the Bushen Kaixuan Tongluo Formula affects DKD via the cAMP pathway remains limited.

In this study, db/db mice were used to simulate human DKD. The Cistanche-based formula significantly improved renal function, general condition, and metabolic disorders in mice, with favorable liver safety profiles. Histopathology showed reduced glomerular basement membrane thickening, mesangial expansion, and interstitial collagen deposition - hallmark DKD lesions.

At the molecular level, the formula upregulated mRNA and protein expression of PKA, CREB, and Bcl-2, enhancing phosphorylation of PKA and CREB, thereby activating the cAMP pathway, improving antioxidant capacity, and restoring filtration barrier function.

Conclusion

The Cistanche-based Bushen Kaixuan Tongluo Formula:

Improves liver and kidney function

Corrects glucose and lipid metabolic disorders

Activates the cAMP/PKA/CREB signaling pathway

Reduces renal fibrosis and oxidative stress

Provides renal protection in DKD

These findings provide a pharmacological basis for its clinical application and support targeting the cAMP pathway as a potential strategy for DKD treatment. Future research should explore downstream molecules such as BDNF to further elucidate the mechanism.