Wentong Huoxue Cream alleviating diabetic peripheral neuropathy by inhibiting the mTOR/4E-BP1 pathway

doi:10.11958/20253618

Abstract

Abstract:

Objective To investigate whether the Wentong Huoxue Cream affected Schwann cells (SC) by regulating the mammalian target of rapamycin (mTOR)/eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) signaling pathway, thereby alleviating sciatic nerve injury in diabetic peripheral neuropathy (DPN) model rats. Methods Sixty 8-week-old male SD rats were randomly divided into the normal group (10 rats) and the model group (50 rats). The model group was established by intraperitoneal injection of streptozotocin (STZ) combined with high-sugar and high-fat diet. The rats in the model group were randomly divided into 5 groups: the model group, the high-dose Wentong Huoxue Cream group, the low-dose Wentong Huoxue Cream group, the matrix group (blank cream) and the metformin group (positive control). After 4 weeks of continuous intervention, the latency of hot tail flicking of rats was detected. The pathological morphology of the sciatic nerve was observed by HE staining. The ultrastructure of myelin sheath and autophagosomes of Schwann cells were observed by transmission electron microscopy. The protein expression levels of mTOR, phosphorylated mTOR (p-mTOR), 4E-BP1, phosphorylated 4E-BP1 (p-4E-BP1) and microtubule-associated protein 1 light chain 3 (LC3-Ⅱ) in sciatic nerve were detected by Western blot assay. The mRNA expressions of mTOR, 4E-BP1 and LC3-Ⅱwere detected by real-time fluorescence quantitative PCR. Results Compared with the model group, the latency of tail flicking was significantly shortened in the Wentong Huoxue Cream groups (P<0.05), the nerve pathological damage and ultrastructural destruction were significantly alleviated and the number of autophagosomes in Schwann cells decreased. Western blot assay and PCR results showed that the protein or mRNA expression levels of p-mTOR, mTOR, p-4E-BP1, 4E-BP1 and LC3-Ⅱwere lower in the Wentong Huoxue Cream groups than those in the model group (P<0.05), and the improvement effect was more significant in the high-dose Wentong Huoxue Cream group than that of the low-dose Wentong Huoxue Cream group. Conclusion Wentong Huoxue Cream can significantly improve neuropathic hyperalgesia and myelin pathological damage in DPN rats. The mechanism may be related to the inhibition of excessive activation of the mTOR/4E-BP1 signaling pathway and the down-regulation of autophagy-related protein LC3-Ⅱ level.

Key words: diabetic neuropathies, TOR serine-threonine kinases, eukaryotic initiation factor-4E, Schwann cells, sciatic nerve, autophagy, Wentong Huoxue Cream

CLC Number:

R587.25

Figures/Tables 7

References 18

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基因名称	引物序列（5′→3′）	产物大小/bp
mTOR	上游：GTGTGGCAAGAGCGGCAGAC 下游：TGTTGGCAGAGGATGGTCAAGTTG	97
4E-BP1	上游：CCCAAAGGACCTGCCAACCATTC 下游：CACCACCTGCCCGCTTATCTTC	116
LC3-Ⅱ	上游：CCAGGACAAGCAGGCAGATGAAG 下游：CAGGCTTTCGTCTCTTCCACCATC	149
β-actin	上游：CCCATCTATGAGGGTTACGC 下游：TTTAAATGTCACGCACGATTTC	151

基因名称	引物序列（5′→3′）	产物大小/bp
mTOR	上游：GTGTGGCAAGAGCGGCAGAC 下游：TGTTGGCAGAGGATGGTCAAGTTG	97
4E-BP1	上游：CCCAAAGGACCTGCCAACCATTC 下游：CACCACCTGCCCGCTTATCTTC	116
LC3-Ⅱ	上游：CCAGGACAAGCAGGCAGATGAAG 下游：CAGGCTTTCGTCTCTTCCACCATC	149
β-actin	上游：CCCATCTATGAGGGTTACGC 下游：TTTAAATGTCACGCACGATTTC	151

组别	血糖/（mmol/L）		体质量/g		热甩尾潜伏期/s
组别	治疗前	治疗后	治疗前	治疗后	热甩尾潜伏期/s
正常组	5.20±0.27	5.55±0.40	506.84±39.50	584.61±32.58	3.98±1.15
模型组	29.85±2.69^a	31.30±1.10^a	374.04±65.64^a	363.96±37.92^a	9.16±2.22^a
低剂量组	30.95±2.97^a	28.35±1.87^a	388.58±42.77^a	358.80±51.64^a	7.14±2.40^ab
高剂量组	29.85±2.07^a	31.61±1.60^a	375.33±57.35^a	347.40±51.54^a	5.02±1.49^abc
基质组	28.25±1.87^a	33.32±1.77^a	374.04±65.64^a	363.96±37.92^a	10.15±3.23^a
二甲双胍组	28.25±2.60^a	7.05±0.90^b	399.56±56.03^a	378.60±44.97^a	6.41±1.66^ab
F	75.130^＊＊	106.876^＊＊	202.301^＊＊	198.103^＊＊	167.504^＊＊

组别	血糖/（mmol/L）		体质量/g		热甩尾潜伏期/s
组别	治疗前	治疗后	治疗前	治疗后	热甩尾潜伏期/s
正常组	5.20±0.27	5.55±0.40	506.84±39.50	584.61±32.58	3.98±1.15
模型组	29.85±2.69^a	31.30±1.10^a	374.04±65.64^a	363.96±37.92^a	9.16±2.22^a
低剂量组	30.95±2.97^a	28.35±1.87^a	388.58±42.77^a	358.80±51.64^a	7.14±2.40^ab
高剂量组	29.85±2.07^a	31.61±1.60^a	375.33±57.35^a	347.40±51.54^a	5.02±1.49^abc
基质组	28.25±1.87^a	33.32±1.77^a	374.04±65.64^a	363.96±37.92^a	10.15±3.23^a
二甲双胍组	28.25±2.60^a	7.05±0.90^b	399.56±56.03^a	378.60±44.97^a	6.41±1.66^ab
F	75.130^＊＊	106.876^＊＊	202.301^＊＊	198.103^＊＊	167.504^＊＊

组别		mTOR		p-mTOR
正常组		0.46±0.02		1.32±0.09
模型组		0.54±0.03^a		2.44±0.32^a
低剂量组		0.45±0.04^b		1.36±0.08^b
高剂量组		0.29±0.04^bc		1.13±0.04^bc
基质组		0.27±0.04^b		1.17±0.24^b
二甲双胍组		0.42±0.06^b		1.27±0.09^b
F		72.265^＊＊		82.863^＊＊
组别	4E-BP1		p-4E-BP1		LC3-Ⅱ
正常组	0.53±0.09		0.12±0.02		0.42±0.04
模型组	1.27±0.17^a		1.29±0.17^a		1.27±0.21^a
低剂量组	0.31±0.05^b		1.17±0.13^b		0.99±0.24^b
高剂量组	1.16±0.13^bc		0.31±0.05^bc		0.56±0.11^bc
基质组	0.41±0.08^b		0.40±0.08^b		0.43±0.10^b
二甲双胍组	0.12±0.02^b		0.53±0.09^b		0.64±0.09^b
F	219.903^＊＊		233.692^＊＊		54.592^＊＊