STZ (60 mg/kg) can successfully induce diabetic peripheral neuropathy in rat

To evaluate the effect of Zhenzhu Tongluo pills on diabetic peripheral neuropathy, a rat model of diabetic peripheral neuropathy was induced by a single intrapitoneal injection of STZ (60 mg/kg), and verified by detecting fasting blood glucose, body weight, mechanical pain and heat pain.

Persistent reduced body weight gain and increased blood glucose are the significant physiological features of STZ rats

Compared with the Control group, fasting glucose levels in the STZ group were significantly increased at day 3, day 7, day 10, day 15, day 20 and day 30 after injection (Fig. 2A, n = 6, two-way ANOVA, ***p < 0.001 vs Control group). In contrast, the STZ group had significantly lower body weight at day 7, day 10, day 15, day 20, and day 30 after injection compared to the Control group (Fig. 2B, n = 6, two-way ANOVA, ***p < 0.001 vs Control group). Since high blood glucose levels and loss of weight gain are prominent features of diabetes, the above results indicate that the rat model of hyperglycemia has been successfully constructed from physiological indicators.

Fig. 2

Single intraperitoneal injection of STZ can successfully induce diabetic peripheral neuropathy in a rat model. A Blood glucose level of STZ group was significantly increased compared with Control group (n = 6; ***p < 0.001 vs Control group); B body weight of STZ group was significantly decreased compared with Control group (n = 6; ***p < 0.001 vs Control group); C mechanical withdrawal threshold of rats in STZ group was significantly lower than that in Control group (n = 6; ***p < 0.001 vs Control group); D paw withdrawal latency of rats in STZ group was significantly decreased compared with Control group (n = 6; ***p < 0.001 vs Control group)

Mechanical and thermal hyperalgesia of STZ rats suggest the success of STZ-induced diabetic peripheral neuropathy in rats

In addition to conventional physiological indicators, mechanical withdrawal threshold and thermal withdrawal latency were evaluated before STZ injection and at 3 days, 7 days, 10 days, 15 days, 20 days and 30 days after STZ injection. We found that the mechanical withdrawal threshold and thermal withdrawal latency were significantly lower in the STZ group at day 7, day 10, day 15, day 20, and day 30 after injection than that in the Control group (Fig. 2C, D, n = 6, two-way ANOVA, ***p < 0.001 vs Control group), showing significant hyperalgesia. These results confirm the successful construction of diabetic peripheral neuropathy rat model.

Zhenzhu Tongluo pills can effectively improve the reduced body weight gain and hyperalgesia of diabetic peripheral neuropathy ratsZhenzhu Tongluo pills can effectively improve the reduced body weight gain of diabetic peripheral neuropathy rats, but has no significant effect on the increased blood glucose

While exploring the neuroprotective effect of Zhenzhu Tongluo pill on diabetic peripheral neuropathy rats, our study also focused on its influence on physiological indexes of diabetic peripheral neuropathy rats. Compared with STZ group, different doses of Zhenzhu Tongluo pills had no significant effect on the increased blood glucose induced by STZ (Fig. 3A). On the other hand, different doses of Zhenzhu Tongluo pills can significantly improve the decreased body weight gain of diabetic peripheral neuropathy rats at different time points. Compared with STZ group, STZ + PTP (50 mg/kg) group showed increased body weight gain at Day 30 and 40 after STZ injection (Fig. 3B, n = 8, two-way ANOVA, *p < 0.05, **p < 0.01 vs STZ group). STZ + PTP (100 mg/kg) group showed significant body weight gain from Day 20 after STZ injection (Fig. 3B, n = 8, two-way ANOVA, #p < 0.05, ###p < 0.001 vs STZ group). STZ + PTP (200 mg/kg) and STZ + PTP (400 mg/kg) groups showed significant body weight gain from Day 15 after STZ injection (Fig. 3B, n = 8, two-way ANOVA, &&&p < 0.001: STZ + PTP (200 mg/kg) vs STZ group; $$$p < 0.001: STZ + PTP (400 mg/kg) vs STZ group. These results suggest that Zhenzhu Tongluo pills can improve part physiological indexes of STZ-induced diabetic peripheral neuropathy rats.

Fig. 3

Zhenzhu Tongluo pills can significantly improve the physiological indexes and sensory dysfunction in diabetic peripheral neuropathy rats. A Zhenzhu Tongluo pills has no significant improvement effect on blood glucose level in diabetic peripheral neuropathy rats; B Zhenzhu Tongluo pills can improve the decreased body weight gain in diabetic peripheral neuropathy rats (n = 8, *p < 0.05, **p < 0.01: STZ + PTP (50 mg/kg) vs STZ group; #p < 0.05, ###p < 0.001: STZ + PTP (100 mg/kg) vs STZ group; &&&p < 0.001: STZ + PTP (200 mg/kg) vs STZ group; $$$p < 0.001: STZ + PTP (400 mg/kg) vs STZ group); C Zhenzhu Tongluo pills significantly improved the mechanical allodynia of diabetic peripheral neuropathy rats (n = 8, ##p < 0.01: STZ + PTP (100 mg/kg) vs STZ group; &p < 0.05, &&p < 0.001: STZ + PTP (200 mg/kg) vs STZ group; $$p < 0.01, $$$p < 0.001: STZ + PTP (400 mg/kg) vs STZ group); D thermal hyperalgesia of diabetic peripheral neuropathy rats was significantly relieved by Zhenzhu Tongluo pills (n = 8; &&&p < 0.001: STZ + PTP (200 mg/kg) vs STZ group; $$p < 0.01, $$$p < 0.001: STZ + PTP (400 mg/kg) vs STZ group)

Zhenzhu Tongluo pills can effectively alleviate mechanical and thermal hyperalgesia in STZ-induced diabetic peripheral neuropathy rats

Neuroprotective function of Zhenzhu Tongluo pills was studied by evaluating its effect on pain sensitization in STZ-induced diabetic peripheral neuropathy rats. For mechanical pain, we found that different doses of Zhenzhu Tongluo pills significantly improved mechanical pain sensitization in STZ-induced rats. Compared with STZ group, STZ + PTP (100 mg/kg) group significantly improved mechanical pain sensitization after Day 20 of STZ injection (Fig. 3C, n = 8, two-way ANOVA, ##p < 0.01 vs STZ group). STZ + PTP (200 mg/kg) group and STZ + PTP (400 mg/kg) group showed relief on mechanical pain sensitization after Day 15 of STZ injection (Fig. 3C, n = 8, two-way ANOVA, &p < 0.001, &&&p < 0.001: STZ + PTP (200 mg/kg) vs STZ group; $$p < 0.01, $$$p < 0.001: STZ + PTP (400 mg/kg) vs STZ group). Similarly, we found that high doses of Zhenzhu Tongluo pills (200 mg/kg and 400 mg/kg) significantly improved thermal hyperalgesia in STZ-induced rats (Fig. 3D, n = 8, two-way ANOVA, &&&p < 0.001: STZ + PTP (200 mg/kg) vs STZ group; $$p < 0.01, $$$p < 0.001: STZ + PTP (400 mg/kg) vs STZ group). The above results indicated that Zhenzhu Tongluo pills had a significant improvement on hyperalgesia in diabetic peripheral neuropathy rats, which suggested that Zhenzhu Tongluo pills has a better neuroprotective function in behavioral performance.

Zhenzhu Tongluo pills can effectively improve the sensory conduction of sciatic nerve in diabetic peripheral neuropathy rats

In fact, abnormal sensory conduction function of sciatic nerve is one of the important features of diabetic peripheral neuropathy rats. By examining the conduction velocity of sensory nerve under different treatment factors, we found that the conduction velocity of sensory nerve in the STZ group was significantly reduced compared with the Control group (Fig. 4, n = 6, one-way ANOVA, ***p < 0.001 vs Control group). After treatment with Zhenzhu Tongluo pills, we found that high-dose Zhenzhu Tongluo pills (200 mg/kg and 400 mg/kg) significantly improved the reduced sensory nerve conduction velocity in diabetic rats (Fig. 4, n = 6, one-way ANOVA, #p < 0.001 vs STZ group). The above results confirm the neuroprotective effect of Zhenzhu Tongluo pills functionally.

Fig. 4

Zhenzhu Tongluo pills can significantly improve the abnormal sensory nerve conduction function in diabetic peripheral neuropathy rats. (n = 6, ***p < 0.001 vs Control group; #p < 0.05 vs STZ group)

Zhenzhu Tongluo pills can improve the high expression of inflammatory factors in DRG of diabetic peripheral neuropathy ratsZhenzhu Tongluo pills reduce the mRNA expression of TNF-α, IL-1β and IL-6 in DRG of diabetic peripheral neuropathy rats

Excessive inflammatory response is associated with the occurrence and development of many diseases. Compared with normal rats, the high expression of pro-inflammatory factors in various tissues of diabetic peripheral neuropathy rats is one of the key factors of pain sensitization. In this study, by constructing an STZ-induced diabetic peripheral neuropathy rat model, we found that the mRNA expression of TNF-α, IL-1β and IL-6 in DRG of STZ rats was significantly increased compared with that in the Control group (Fig. 5, n = 4, one-way ANOVA, **p < 0.01, ***p < 0.001 vs Control group). This confirmed the increased expression of inflammatory factors in DRG of diabetic peripheral neuropathy rats at mRNA level. By treating diabetic peripheral neuropathy rats with different doses of Zhenzhu Tongluo pills, we found that different doses of Zhenzhu Tongluo pills significantly improved the high expression of inflammatory factors at mRNA level in DRG of STZ rats (Fig. 5, n = 4, one-way ANOVA, #p < 0.05, ##p < 0.01, ###p < 0.001 vs STZ group).

Fig. 5

Zhenzhu Tongluo pills can significantly improve the increased expression of inflammatory factors at the mRNA level in DRG of diabetic peripheral neuropathy rats. A Zhenzhu Tongluo pills can significantly improve the expression of TNF-α on mRNA level in DRG of diabetic peripheral neuropathy rats (n = 4, ***p < 0.001 vs Control group; #p < 0.05, ###p < 0.001 vs STZ group); B Zhenzhu Tongluo pills could significantly improve the expression of IL-1β on mRNA level in DRG of diabetic peripheral neuropathy rats (n = 4, ***p < 0.001 vs Control group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs STZ group); C Zhenzhu Tongluo pills could significantly improve the expression of IL-6 on mRNA level in DRG of diabetic peripheral neuropathy rats (n = 4, **p < 0.01 vs Control group; #p < 0.05 vs STZ group)

Zhenzhu Tongluo pills reduce the protein expression of TNF-α, IL-1β and IL-6 in DRG of diabetic peripheral neuropathy rats

After determining the expression of inflammatory factors at the mRNA level, we then detected the expression of TNF-α, IL-1β and IL-6 at the protein level by ELISA. Compared with Control group, the expression of inflammatory cytokines TNF-α, IL-1β and IL-6 in DRG in STZ group was significantly increased at the protein level (Fig. 6, n = 6, one-way ANOVA, ***p < 0.001 vs Control group). After treatment with different doses of Zhenzhu Tongluo pills, we found that the expression of TNF-α, IL-1β and IL-6 in DRG in the treatment group was significantly reduced compared with that in the STZ group (Fig. 6, n = 6, one-way ANOVA, #p < 0.05, ##p < 0.01). ###p < 0.001 vs STZ group).

Fig. 6

Zhenzhu Tongluo pills can significantly improve the increased expression of inflammatory factors at the protein level in DRG of diabetic peripheral neuropathy rats. A Zhenzhu Tongluo pills can significantly improve the expression of TNF-α in the protein level of DRG in diabetic peripheral neuropathy rats (n = 6, ***p < 0.001 vs Control group; ###p < 0.001 vs STZ group); B Zhenzhu Tongluo pills can significantly improve the expression of IL-1β in DRG of diabetic peripheral neuropathy rats (n = 6, ***p < 0.001 vs Control group; ##p < 0.01, ###p < 0.001 vs STZ group); C Zhenzhu Tongluo pills can significantly improve the expression of IL-6 in DRG of diabetic peripheral neuropathy rats (n = 6, ***p < 0.001 vs Control group; #p < 0.05 vs STZ group)

The above results confirmed that Zhenzhu Tongluo pills has a significant anti-inflammatory effect at the mRNA level and protein level, and it can effectively improve the increased inflammatory response in the DRG of STZ-induced diabetic peripheral neuropathy rats.

Zhenzhu Tongluo pills modulate the expression of several key signaling molecules in DRG of diabetic peripheral neuropathy ratsZhenzhu Tongluo pills can regulate the expression of PI3K–AKT pathway protein in DRG of diabetic peripheral neuropathy rats

The expression changes of PI3K, p-PI3K, AKT and p-AKT in DRG of rats in different groups were detected by WB. Our results showed that no significant changes of total PI3K and AKT were found among different groups (Fig. 7A, B, D), but the expression of p-PI3K and p-AKT were significantly lower (Fig. 7A, C, E, n = 3, one-way ANOVA, **p < 0.01, ***p < 0.001 vs Control group) than Control group; And the expression of p-PI3K and p-Akt were increased to varying degrees after treatment with different doses of Zhenzhu Tongluo pills (Fig. 7A, C, E, n = 3, one-way ANOVA, #p < 0.05 vs STZ group). These results suggest that Zhenzhu Tongluo pills can regulate the expression of PI3K–AKT pathway protein molecules.

Fig. 7

Effect of Zhenzhu Tongluo pills on the expression of protein molecules in PI3K–AKT signaling pathway in DRG of diabetic peripheral neuropathy rats. A, B There was no significant change in the expression of total PI3K protein in DRG among all groups (n = 3); A, C Zhenzhu Tongluo pills could significantly improve p-PI3K expression in DRG of diabetic peripheral neuropathy rats (n = 3, ***p < 0.001 vs Control group; #p < 0.05 vs STZ group); A, D There was no significant change in the expression of total AKT in DRG among all groups (n = 3). A, E Zhenzhu Tongluo pills could significantly improve the reduced expression of p-Akt in DRG of diabetic peripheral neuropathy rats (n = 3, **p < 0.01 vs Control group; #p < 0.05 vs STZ group)

Immunohistochemical staining showed that Zhenzhu Tongluo pills could significantly improve the low expression of p-AKT in situ in DRG of STZ rats

The expression of p-AKT in different groups of DRG was detected in situ by immunohistochemistry. Compared with the Control group, the expression of p-Akt in STZ group was significantly reduced (Fig. 8A, B, n = 6, one-way ANOVA, ***p < 0.001 vs Control group). After treatment with different doses of Zhenzhu Tongluo pills, the expression of p-Akt was increased to varying degrees (Fig. 8A, B, n = 6, one-way ANOVA, #p < 0.05, ##p < 0.01 vs STZ group).

Fig. 8

Immunohistochemical staining showed that Zhenzhu Tongluo pills could improve the decreased expression of p-Akt in DRG of diabetic peripheral neuropathy rats (n = 3, ***p < 0.001 vs Control group; #p < 0.05, ##p < 0.01 vs STZ group; Scale bar = 20 μm)

Zhenzhu Tongluo pills can regulate the expression of NF-κB p65 and p38 MAPK protein in DRG of diabetic peripheral neuropathy rats

In addition to the PI3K–AKT signaling pathway, we also detected the expression of molecular proteins related to the MAPK and NF-κB signaling pathways. Compared with the Control group, total protein expression of NF-κB p65 and p38 MAPK in each group had no significant change (Fig. 9A, B, D, n = 3), but p–p38 MAPK and p-NF-κB p65 expression were significantly increased (Fig. 9A, C, E, n = 3, one-way ANOVA, **p < 0.01, ***p < 0.001 vs Control group); The expression of p–p38 MAPK and p-NF-κB p65 decreased to varying degrees after treatment with different doses of Zhenzhu Tongluo pills (Fig. 9A, C, E, n = 3, one-way ANOVA, #p < 0.05, ##p < 0.01 vs STZ group).

Fig. 9

Effect of Zhenzhu Tongluo pills on the expression of NF-κB p65 and p38 MAPK protein in DRG of diabetic peripheral neuropathy rats. A, B There was no significant change in the expression of p38 MAPK protein in DRG among all groups (n = 3). A, C Zhenzhu Tongluo pills can significantly improve the increased expression of p-P38 MAPK in DRG of diabetic peripheral neuropathy rats (n = 3, ***p < 0.001 vs Control group; #p < 0.05, ##p < 0.01 vs STZ group); A, D There was no significant change in the expression of total NF-κB p65 protein in DRG among all groups (n = 3). A, E Zhenzhu Tongluo pills can significantly improve the increased expression of p-NF-κB p65 in DRG of diabetic peripheral neuropathy rats (n = 3, **p < 0.01 vs Control group; #p < 0.05 vs STZ group)

Zhenzhu Tongluo pills can regulate the expression of ERK and JNK in DRG of diabetic peripheral neuropathy rats

Compared with the Control group, the total protein of JNK and ERK in all groups were not significantly changed (Fig. 10A, B, D, n = 3), while the expression of p-ERK and p-JNK in STZ group were significantly increased (Fig. 10A, C, E, n = 3, one-way ANOVA, *p < 0.05, ***p < 0.001 vs Control group). The expression of p-ERK and p-JNK were decreased to varying degrees after treatment with different doses of Zhenzhu Tongluo pills (Fig. 10A, C, E, n = 3, one-way ANOVA, #p < 0.05, ##p < 0.01 vs STZ group). In addition, different doses of Zhenzhu Tongluo pills can also improve the expression of c-Fos in DRG of diabetic rats to varying degrees (Fig. 10A, F, n = 3, one-way ANOVA, ***p < 0.001 vs Control group; #p < 0.05, ##p < 0.01 vs STZ group).

Fig. 10

Effect of Zhenzhu Tongluo pills on the expression of JNK and ERK proteins in DRG of diabetic peripheral neuropathy rats. A, B There was no significant change in the expression of p38 MAPK protein in DRG among all groups (n = 3). A, C Zhenzhu Tongluo pills could significantly improve the increased expression of p-JNK in DRG of diabetic peripheral neuropathy rats (n = 3, *p < 0.05 vs Control group; #p < 0.05 vs STZ group); A, D There was no significant change in the expression of total ERK protein in DRG among all groups (n = 3). A, E Zhenzhu Tongluo pills could significantly improve the increased expression of p-ERK in DRG of diabetic peripheral neuropathy rats (n = 3, ***p < 0.001 vs Control group; #p < 0.05, ##p < 0.01 vs STZ group); A, F: Zhenzhu Tongluo pills could significantly improve the increased expression of c-FOS in DRG of diabetic peripheral neuropathy rats (n = 3, ***p < 0.001 vs Control group; #p < 0.05, ##p < 0.01 vs STZ group)

Zhenzhu Tongluo pills can relieve the increase of apoptosis in DRG of diabetic peripheral neuropathy rats

The above studies showed that the dose of Zhenzhu Tongluo pills (200 mg/kg) was effective and stable, so the molecular index of this dose was selected to verify the effectiveness of Zhenzhu Tongluo pills. TUNEL staining was used to observe the apoptosis of DRG cells in different groups. Compared with Control group, the TUNEL positive staining rate in DRG of rats in STZ group was significantly increased (Fig. 11A, B, n = 8, one-way ANOVA, ***p < 0.001 vs Control group). Compared with STZ group, Zhenzhu Tongluo pills treatment (STZ + PTP; 200 mg/kg) significantly decreased the positive rate of TUNEL staining in DRG (Fig. 11A, B, n = 8, one-way ANOVA, ###p < 0.001 vs STZ group). The above results indicate that Zhenzhu Tongluo pills can play an effective anti-apoptotic role.

Fig. 11

Zhenzhu Tongluo pills can significantly improve the increased apoptosis in DRG of diabetic peripheral neuropathy rats. A TUNEL staining diagram (Scale bar = 20 μm); B Zhenzhu Tongluo pills could significantly improve the increased apoptosis in DRG of diabetic peripheral neuropathy rats (n = 8, ***p < 0.001 vs Control group; ###p < 0.001 vs STZ group)

Zhenzhu Tongluo pills can relieve sciatic nerve injury in diabetic peripheral neuropathy rats

One of the reasons for hyperalgesia in diabetic peripheral neuropathy rats is the obvious injury of sciatic nerve. The above research results indicate that relieving inflammation and modulating the expression of various signaling pathway protein molecules are the possible mechanisms of relieving pain by Zhenzhu Tongluo pills in diabetic peripheral neuropathy rats. However, does Zhenzhu Tongluo pills have a protective effect on sciatic nerve injury in STZ rats? We do not know. Therefore, in this part of the study, HE staining and electron microscopy were used to observe the morphological characteristics of sciatic nerves in different groups.

HE staining showed that Zhenzhu Tongluo pills could alleviate sciatic nerve structural injury in diabetic peripheral neuropathy rats

HE staining was performed on the longitudinal and transverse sections of sciatic nerves of rats in different groups to observe the tissue morphology. In Control group, the nerve fibers of rats were normal in size, dense and uniform in distribution, uniform in myelin staining, and axons in the myelin were clear without swelling or atrophy (Fig. 12). In STZ group, the nerve fibers were loosely arranged, myelin edema was uneven, and even demyelination occurred, and axons were significantly changed (Fig. 12). After treatment with Zhenzhu Tongluo pills, the pathological damage of nerve fibers was significantly improved, and the arrangement of nerve fibers was more orderly and the staining density was uniform (Fig. 12).

Fig. 12

HE staining showed that Zhenzhu Tongluo pills could significantly improve sciatic nerve injury in diabetic peripheral neuropathy rats (Scale bar = 20 μm)

Electron microscope showed that Zhenzhu Tongluo pills could alleviate sciatic nerve structural injury in diabetic peripheral neuropathy rats

Transmission electron microscopy (TEM) was used to observe the ultrastructure of the transverse section of sciatic nerve in different groups. In Control group, the myelin sheath structure of myelinated nerve fibers was complete, dense and neatly arranged, and the myelin plate presented a concentric circular appearance with intact axons (Fig. 13). In STZ group, myelin lamellar separation, axon atrophy and even demyelination of myelinated nerve fibers occurred (Fig. 13). Zhenzhu Tongluo pills treatment (STZ + PTP; 200 mg/kg) significantly reduced demyelination and axon damage, and enhanced myelin regeneration, showed almost well-organized myelin nerve fibers (Fig. 13).

Fig. 13

Electron microscope showed that Zhenzhu Tongluo pills could significantly improve sciatic nerve injury in diabetic peripheral neuropathy rats (Scale bar = 1 μm)