Traditional Chinese medicine composition for preventing and / or treating diabetic peripheral neuropathy and preparation method thereof

Abstract

The invention discloses a traditional Chinese medicine composition for preventing and / or treating diabetic peripheral neuropathy and a preparation method thereof. The traditional Chinese medicine composition is prepared from the following raw materials in parts by mass: 6-40 parts of angelica sinensis, 9-120 parts of astragalus membranaceus, 0.5-15 parts of pseudo-ginseng, 3-30 parts of sappan wood, 3-30 parts of cassia twig, 3-100 parts of ligusticum wallichii, 5-120 parts of radix paeoniae alba, 3-200 parts of black prepared common monkshood daughter root and 9-70 parts of ramulus mori. A preparation method comprises the following steps: soaking the raw material medicines in cold water for 30 minutes, adding water to exceed the surface of the medicines by 2-3cm, decocting for the first time, boiling with big fire (strong fire), then keeping boiling for 25-35 minutes with soft fire (slow fire) until the medicinal liquid is left for 200ml, pouring out, decocting for the second time, adding water again until the medicinal liquid just covers the surface of the medicines, boiling with big fire, then keeping boiling for 25-35 minutes with soft fire (slow fire) until the medicinal liquid is left for 200ml, pouring out, and filtering to obtain a finished product. And mixing the two decoctions (the granules can be taken after being mixed with warm water). The traditional Chinese medicine composition is scientific and reasonable in compatibility, rich in raw materials, convenient to take and good in effect, has the effects of tonifying qi, activating blood, dispersing blood stasis and dredging collaterals, can be effectively used for treating DPN patients, and is an innovation of DPN treatment medicines.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to a traditional Chinese medicine composition for preventing and / or treating diabetic peripheral neuropathy and a preparation method thereof. Background Art

[0002] Diabetic peripheral neuropathy (DPN) is one of the most common chronic complications of diabetes. Clinical symptoms include "glove-like" numbness, paresthesias, intractable neuropathic pain, and ulcers. Western medicine primarily treats DPN by alleviating clinical symptoms, and no specific medication is currently available. Traditional Chinese medicine (TCM) offers unique advantages in symptom relief, neuroprotection, and repair.

[0003] Traditional Chinese Medicine considers DPN to be part of the "deficiency and damage" phase of diabetes. Prolonged thirst leads to a deficiency of Qi, blood, and Yin and Yang. This long-term condition affects the collaterals. Qi is the leader of blood, and qi deficiency leads to blood stasis, ultimately leading to impaired blood circulation and vascular stasis. As the Jingyue Complete Works states, "The qi and blood of ordinary people are like springs; abundant flow smoothly, while deficient flow stagnates." Therefore, "those with sufficient qi and blood deficiency experience no stagnation, and those with deficiency experience no stagnation." Treatment should prioritize: tonifying qi to disperse blood stasis, tonifying qi without stagnation. Resolving stasis promotes the natural flow of qi and blood, allowing blood to circulate without harming vital energy. When qi is energized, stasis is eliminated, and the collaterals are unblocked, the patient's symptoms will improve. Treatment should prioritize tonifying qi, activating blood circulation, and resolving stasis to unclog the collaterals. Summary of the Invention

[0004] In view of the defects of the existing technology, the purpose of the present invention is to provide a traditional Chinese medicine composition for preventing and / or treating diabetic peripheral neuropathy (DPN), which can effectively solve the medication problem of DPN.

[0005] To achieve the above object, the present invention is implemented through the following technical solutions: A traditional Chinese medicine composition for preventing and / or treating diabetic peripheral neuropathy (DPN), prepared from the following raw materials in parts by weight: 6-40 parts of angelica sinensis, 9-120 parts of astragalus, 0.5-15 parts of Panax notoginseng, 3-30 parts of sappan wood, 3-30 parts of cassia twig, 3-100 parts of Chuanxiong, 5-120 parts of white peony root, 3-200 parts of Heishunpian, and 9-70 parts of mulberry branch.

[0006] Preferably, the traditional Chinese medicine composition is made of the following raw materials in parts by mass: 6-20 parts of angelica, 9-30 parts of astragalus, 0.5-9 parts of Panax notoginseng, 3-27 parts of Sappan wood, 3-27 parts of cinnamon twig, 3-30 parts of Chuanxiong, 6-20 parts of white peony root, 3-20 parts of Heishunpian, and 9-30 parts of mulberry branch.

[0007] More preferably, the Chinese medicine composition is made from the following raw materials in parts by mass: 9 parts of angelica, 15 parts of astragalus, 3 parts of Panax notoginseng, 9 parts of Sappan wood, 9 parts of cinnamon twig, 15 parts of Chuanxiong, 9 parts of white peony root, 3 parts of Heishunpian, and 15 parts of mulberry branch.

[0008] The invention has scientific and reasonable compatibility, rich raw materials, convenient administration, good effect, and can play the effects of invigorating qi and activating blood circulation, removing blood stasis and unblocking meridians. It can be effectively used to treat patients with DPN and is an innovation in the treatment of DPN drugs.

[0009] The raw materials used to make the Chinese medicine composition of the present invention are proportioned according to parts by mass. During production, they can be increased or decreased according to the corresponding proportions. For example, large-scale production can be measured in kilograms or tons, and small-scale production can be measured in grams. The weight can be increased or decreased, but the mass ratio of the raw materials between the components remains unchanged.

[0010] The present invention also provides a preparation method of the traditional Chinese medicine composition.

[0011] The preparation method of the traditional Chinese medicine composition provided by the present invention comprises the following steps: mixing the raw materials, adding water and boiling for extraction, and collecting the supernatant after the extraction is completed to obtain the traditional Chinese medicine composition.

[0012] Furthermore, the above raw materials are soaked in cold water for 30 minutes, and the amount of water added exceeds the medicine surface by 2-3 cm. The first decoction is first boiled with high heat (high heat), and then kept boiling with low heat (low heat) for 25-35 minutes, until the medicine liquid is 30-50% of the initial water amount, poured out, and the second decoction is added with water again, just enough to soak the medicine surface, boiled with high heat, and then kept boiling with low heat (low heat) for 25-35 minutes, until the medicine liquid is 30-50% of the initial water amount, poured out, and the two decoctions are mixed to obtain the product.

[0013] Furthermore, the method further comprises the step of freeze-drying the obtained supernatant to obtain freeze-dried powder.

[0014] At the same time, the prevention and / or treatment of diabetic peripheral neuropathy (DPN) prepared with the Chinese medicine composition provided by the present invention as an active ingredient also falls within the protection scope of the present invention.

[0015] The present invention also protects the use of the above-mentioned traditional Chinese medicine composition in the preparation of a medicament for preventing and / or treating diabetic peripheral neuropathy (DPN).

[0016] The present invention also protects a drug for preventing and / or treating diabetic peripheral neuropathy (DPN).

[0017] The active ingredient of the drug for preventing and / or treating diabetic peripheral neuropathy (DPN) includes the above-mentioned traditional Chinese medicine composition.

[0018] Furthermore, the active ingredient may also be only the above-mentioned traditional Chinese medicine composition.

[0019] The drug can be prepared directly from the pharmaceutical composition or by adding pharmaceutically acceptable excipients to form any pharmaceutically acceptable dosage form. Oral preparations, for example, include granules, capsules, tablets, oral liquids, powders, pills, dripping pills, sustained-release preparations, oral liquids, mixtures, and syrups. The various dosage forms of the drug can be prepared according to conventional methods in the pharmaceutical field.

[0020] Furthermore, the pharmaceutical composition or drug is a drug suitable for treating pain, numbness and paresthesia symptoms caused by diabetic peripheral neuropathy.

[0021] Furthermore, the prevention and / or treatment of diabetic peripheral neuropathy (DPN) is embodied in at least one of the following aspects: 1) Reduce pain sensitivity; 2) Increase nerve conduction velocity; 3) Improve the decreased blood perfusion around the foot pad and sciatic nerve; 4) Improve neurological phenotype and restore neuronal function; 5) Can improve sciatic nerve pathological damage; 6) Inhibit inflammatory response; 7) Promoted mitochondrial fusion and inhibited mitochondrial fission by activating the AMPK signaling pathway; 8) Improved STZ-induced sciatic nerve apoptosis in diabetic rats; 9) Improved oxidative stress and apoptosis of Schwann cells (SCs) exposed to high glucose (HG) and alleviated mitochondrial structural and functional damage.

[0022] The angelica in the prescription of the present invention is sweet, pungent and warm in nature, and has the characteristics of promoting blood circulation without consuming blood. It is called "the qi medicine in the blood, the holy medicine in the blood", and is used to treat blood stasis caused by various reasons. Because the pathological changes of blood stasis run through the whole process of diabetes, the blockage of collateral vessels is the key pathogenesis of the occurrence and development of diabetic neuropathy. Therefore, this medicine is used to promote blood circulation and replenish blood, warm the meridians, and is the monarch medicine in the prescription; Astragalus is an important qi-invigorating medicine, sweet in taste and slightly warm in nature, which can greatly replenish vital energy, make qi vigorous and blood circulate, and assist angelica in promoting blood circulation; Panax notoginseng is sweet, slightly bitter and warm in nature, and enters the liver and stomach meridians, has the functions of dispersing blood stasis and stopping bleeding, reducing swelling and alleviating pain, and can resolve blood stasis without hurting new blood, so it is a wonderful product for regulating blood; Sappan wood is sweet, salty, slightly astringent and flat in nature, and enters the heart, liver and spleen meridians, and has the effects of promoting blood circulation, removing blood stasis, reducing swelling and relieving pain; Cassia twig is pungent, sweet and warm to dredge the meridians, travel to the limbs and the skin of the muscles, and can also dredge the meridians and open blockages; Chuanxiong is pungent in taste and warm in nature, and is a qi medicine of the blood. When Qi flows, blood flows, and when blood flows, blood stasis dissipates. The five herbs used together can replenish Qi and activate blood circulation, while also strengthening the main herb's ability to dissolve blood stasis, making them the assistant herbs in the formula. White peony root is sweet, bitter, and sour, and slightly cold in nature. It nourishes blood and regulates menstruation. When combined with astragalus root, it both invigorates Qi and nourishes blood, achieving a method of simultaneously replenishing Qi, blood, and Yin and Yang. When combined with cinnamon twig, it harmonizes the Ying and Wei, complementing each other. Heishun tablets are pungent, warm, and highly hot, and when used together, they can harmonize Yin and Yang, Qi, and blood, serving as adjuvants. Morus alba is neutral in nature, slightly bitter in taste, and enters the liver meridian. It is good at unblocking meridians, reaching the limbs, benefiting joints, and has analgesic properties. In the formula, it can guide the meridians and enable the herbs to reach the meridians throughout the body, serving as the assistant herb. The entire formula works together to replenish Qi, activate blood circulation, dissipate blood stasis, and dredge meridians. It has significant clinical efficacy in treating PDN. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Changes in thermal and mechanical pain thresholds in rats of each group. (A) TWL. (B) MWT. Compared with the control group, #p<0.05, ##p<0.01; compared with the model group, *p<0.05, **p<0.01; compared with the ALA group, △p<0.05, △△p<0.01.

[0024] Figure 2 Comparison of motor and sensory nerve conduction velocity and action potential amplitude in rats among the groups. (A) SNCV. (B) MNCV. (C) SNAP. (D) CMAP. Compared with the CTRL group, #p<0.05, ##p<0.01; compared with the STZ group, *p<0.05, **p<0.01; compared with the ALA group, △p<0.05, △△p<0.01.

[0025] Figure 3Comparison of blood perfusion in the footpad and sciatic nerve of rats in each group. (A, B) Representative images of blood perfusion in the footpad and sciatic nerve of rats in the CTRL, STZ, TBF, and ALA groups. (C, D) Quantitative analysis of blood perfusion. The lowest blood flow is indicated in blue, the highest blood flow in red, and intermediate blood flow in green and yellow. N = 5. Compared with the CTRL group, #p < 0.05, ##p < 0.01; compared with the STZ group, *p < 0.05, **p < 0.01; compared with the ALA group, △p < 0.05, △△p < 0.01.

[0026] Figure 4 H&E staining of rat sciatic nerve. Two different magnifications (200X; 400X) were selected for each group, and the scale bars were 100 μm and 30 μm, respectively.

[0027] Figure 5 LFB staining of rat sciatic nerve (bars: 100 μm and 30 μm).

[0028] Figure 6 Silver staining of rat sciatic nerve (scale bars: 100 μm and 30 μm).

[0029] Figure 7 Nissl staining of SD rats. (A) Representative images of Nissl staining (bar, 30 μm). (B) Quantitative analysis of the number of neurons per unit area. N = 5. Compared with the CTRL group, #p < 0.05, ##p < 0.01; compared with STZ, *p < 0.05, **p < 0.01.

[0030] Figure 8 Transmission electron microscopy (TEM) observations of the ultrastructure of sciatic nerve tissue. (A) Representative images of sciatic nerves obtained by transmission electron microscopy. Panels show cross-sections at 4000x (2 μm) and 8000x (1 μm) magnifications. (B) G-ratio measurements in sciatic nerves. Data are expressed as mean ± SD, n = 3 per group. Compared with the CTRL group, #p < 0.05, ##p < 0.01; compared with the STZ group, *p < 0.05, **p < 0.01; compared with the ALA group, △p < 0.05, △△p < 0.01. A: axon; MS: myelin sheath; N: nucleus; black arrow: mitochondria; Nu: nucleolus.

[0031] Figure 9 The expression levels of IL-6 (A), TNF-a (B), and IL-1β (C) in the serum of rats in each group. N=5. Compared with the CTRL group, # p <0.05, ## p <0.01; compared with STZ,* p<0.05,** p <0.01; compared with the ALA group, p <0.05, △△ p <0.01.

[0032] Figure 10 The expression levels of serum SOD (A) and MDA (B) in rats of each group. N=5. Compared with the CTRL group, # p <0.05,## p <0.01; compared with STZ, * p <0.05, ** p <0.01.

[0033] Figure 11 The apoptosis of rat sciatic nerve was detected by TUNEL method.

[0034] Figure 12 Immunohistochemistry was used to detect changes in S-100β protein levels. (A) Representative images of S-100β protein expression levels in each group. (B) Statistics of the ratio of S-100β protein positive areas. N = 5. Compared with the CTRL group, # p <0.05, ## p <0.01; compared with STZ, * p <0.05, ** p <0.01.

[0035] Figure 13 TBF modulates changes in the AMPK pathway and its downstream mitochondrial dynamics in the sciatic nerve of STZ-induced diabetic rats. (A) Western blot analysis of the expression levels of p-AMPK, AMPK, PGC-1a, p-Drp1, Drp1, Fis1, Mfn1, Mfn2, and Opa1 in the sciatic nerve. (B) Protein quantification results are presented as fold change compared with the CTRL group. N = 5 per group. Compared with the CTRL group, # p <0.05, ## p <0.01; compared with STZ, * p <0.05, ** p <0.01.

[0036] Figure 14 Figure 5. Effects of HG on cell viability of SCs exposed to HG for 24, 48, and 72 hours. Compared with NG, #p<0.05, ##p<0.01; compared with HG, *p<0.05, **p<0.01.

[0037] Figure 15SCs were cultured in HG medium containing different concentrations of TBF (50, 100, 200, 400, 800, and 1000 μg / mL) for 48 h to examine the effect of TBF on cell viability during HG exposure. Compared with NG, #p<0.05, ##p<0.01; compared with HG, *p<0.05, **p<0.01.

[0038] Figure 16 Effects of TBF on ROS levels in Schwann cells from HG-exposed rats. Values ​​represent triplicate experiments and the mean DCF fluorescence intensity fold change relative to the NG group. Data are shown as mean ± SD; compared with NG, # p <0.05,## p <0.01; compared with HG, * p <0.05,** p <0.01; compared with the AICAR group, p <0.05, △△ p <0.01.

[0039] Figure 17 The effect of TBF on MMP in Schwann cells of rats exposed to HG. Data are shown as mean ± SD. Compared with NG, # p <0.05,## p <0.01; compared with HG, * p <0.05,** p <0.01.

[0040] Figure 18 The effect of TBF on the apoptosis of SCs exposed to HG. Data are shown as mean ± SD. Compared with NG, # p <0.05,## p <0.01; compared with HG, * p <0.05,** p <0.01.

[0041] Figure 19 The effect of TBF on SC ATP in HG-exposed SCs. Data are shown as mean ± SD. Compared with NG, # p <0.05,## p <0.01; compared with HG, * p <0.05,** p <0.01.

[0042] Figure 20 The effect of TBF on the expression of MAD and SOD in HG-treated SCs. Data are shown as mean ± SD. Compared with NG, # p <0.05,## p<0.01; compared with HG, * p <0.05,** p <0.01.

[0043] Figure 21 TBF modulates HG-induced changes in the expression levels of proteins involved in the AMPK pathway and its downstream mitochondrial dynamics in SCs. (A) Western blot analysis of the expression levels of p-AMPK, AMPK, PGC-1a, p-Drp1, Drp1, Fis1, Mfn1, Mfn2, and Opa1 in sciatic nerves. (B) Protein quantification results are presented as fold changes compared with the CTRL group. N = 3 per group. Compared with the CTRL group, #p<0.05, ##p<0.01; compared with STZ, *p<0.05, **p<0.01.

[0044] Figure 22 Transmission electron microscopy observation of the effects of TBF on the ultrastructure of mitochondria in HG-treated SCs. M: mitochondria; RER: rough endoplasmic reticulum; N: nucleus. Magnification: 500 nm. DETAILED DESCRIPTION

[0045] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0046] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0047] The Chinese angelica, astragalus, Panax notoginseng, Sappan wood, cinnamon twig, Chuanxiong, white peony root, Heishun Pian, and mulberry twig used in the following examples all comply with the relevant provisions of the Chinese Pharmacopoeia (2025 edition) under the main text of Volume 1. Before adding the materials, the actual ingredients were identified as consistent with their names and met the quality standards.

[0048] Example 1. Preparation of a Traditional Chinese Medicine Composition for Preventing and / or Treating Diabetic Peripheral Neuropathy (DPN) (hereinafter referred to as TBF) 1. Prescription Angelica 9g, Astragalus 15g, Panax notoginseng 3g, Sappan wood 9g, Cinnamon twig 9g Chuanxiong 15g, White Peony 9g, Heishun Pian 3g, Mulberry Branch 15g The total amount of raw herbs in the prescription is 87g.

[0049] 2. Preparation process Soak the nine herbs—astragalus, angelica, Chuanxiong, Sappan wood, mulberry twig, cassia twig, white peony root, Panax notoginseng, and Heishun Pian—in cold water for 30 minutes. Add water to a volume 2-3 cm above the surface of the herbs. For the first decoction, boil over high heat, then simmer over low heat for 25-35 minutes until the liquid is 30-50% of the initial volume (approximately 200 ml). Pour the water out. For the second decoction, add water again, just enough to cover the surface of the herbs. Bring to a boil over high heat, then simmer over low heat for 25-35 minutes until the liquid is 30-50% of the initial volume (approximately 200 ml). Pour the water out. This is the Tangbi formula. To clarify the dose-effect relationship, a freeze-dried powder was prepared for further study. The extract was cooled and centrifuged (8,000 rpm, 10 min), and the supernatant was collected. Finally, a powdered extract of TBF was obtained by freeze-drying to prepare TBF freeze-dried powder.

[0050] Example 2: Animal Experimental Pharmacodynamics Study 1. Experimental Materials A diabetic rat model (DM) was established using intraperitoneal injection of STZ. After successful DM model establishment, TBF and α-lipoic acid (ALA) were administered as pharmacological interventions. During the intervention, the rats' general condition, body weight, and blood glucose levels were monitored, as well as neurological phenotypes including mechanical and thermal pain thresholds and nerve conduction velocity. Changes in sciatic nerve blood flow were also measured. Pathological changes and cellular ultrastructure of the sciatic nerve were also observed to investigate the protective effects of TBF on neurological function and morphology in DM rats.

[0051] 1.1 Experimental Animals Sixty SPF adult male Sprague-Dawley rats, 6 to 8 weeks old, with an initial body weight of 200 ± 20 g, were housed at the Animal Experimental Center of Changchun University of Chinese Medicine, five rats per cage, in a 12-h light / 12-h dark cycle at a temperature of 18–22°C and a relative humidity of approximately 40%–70%.

[0052] 1.2 Experimental drugs The experimental drug used was the TBF lyophilized powder prepared in Example 1.

[0053] Preparation of TBF: Dissolve TBF in sterile PBS to prepare a 10 mg / mL TBF stock solution. Store at -20°C until ready for use. Dilute with culture medium to the desired concentration.

[0054] According to the equivalent dose ratio between humans and animals, the daily dosage required for rats was calculated and divided into low, medium and high dose groups, containing TBF raw drug amounts of 0.28 g / kg, 0.56 g / kg and 1.12 g / kg respectively.

[0055] 2. Experimental Methods 2.1 Animal modeling and grouping SD rats were acclimated to a diet for one month before the experiment. Subsequently, 10 rats were randomly selected as the control group (CTRL) and the remaining 50 rats as the experimental group for model establishment. After a 12-hour fast, the experimental group rats were intraperitoneally injected with 1% STZ solution (65 mg / kg) dissolved in 0.1 M sodium citrate buffer (pH 4.5). Simultaneously, the CTRL group rats were intraperitoneally injected with an equal volume of sodium citrate buffer. After the injection, the rats were inspected for bleeding, and disinfection and hemostasis were promptly performed. Immediately after model establishment, the rats were provided with ample water to prevent hyperosmotic death caused by hyperglycemia. Bedding was changed daily to maintain a clean and tidy living environment.

[0056] Three days after model establishment, fasting plasma glucose (FBS) levels were measured in the rats' tail veins. Ten rats in the experimental group with suboptimal blood glucose levels were excluded based on the results, and the remaining rats were randomly divided into five groups (n = 8 / group). The day the DM model was successfully established was designated day 0, and drug intervention began. The drug intervention regimen was as follows: STZ group: rats with pre-established DPN were gavaged with 1.5 ml of 0.9% saline; TBF group: rats with pre-established DPN were gavaged with TBF containing 0.28 g / kg / day, 0.56 g / kg / day, and 1.12 g / kg / day of TBF; ALA group: rats with pre-established DPN were gavaged with α-lipoic acid (0.054 g / kg / day). The control group received an equal volume of saline. Drug intervention lasted for 12 weeks. All rats were fed a standard diet with ad libitum access to water, and fresh bedding was replaced daily.

[0057] 2.2 Index detection At specified time points, the rats' general condition, blood sugar, body weight, thermal pain threshold, mechanical pain threshold, and nerve conduction velocity were measured. Laser speckle blood flow imaging, sciatic nerve histopathological staining, and sciatic nerve ultrastructure were also used for observation.

[0058] 3. Results 3.1 Effects of TBF freeze-dried powder (Tangbi Fang) on ​​the behavior of diabetic rats After 12 weeks of drug intervention, the hot plate test was used to detect changes in the thermal pain threshold of rats in each group. The results showed that the pain threshold of rats in the STZ group under thermal stimulation was significantly lower than that in the CTRL group (p<0.05), indicating hyperalgesia. The thermal pain threshold of the 1.12 g / kg TBF group and the ALA group was significantly prolonged compared with the STZ group (p<0.05). The mechanical pain threshold of the STZ group was significantly lower than that of the CTRL group (p<0.01). After 12 weeks of TBF and ALA intervention, the mechanical pain threshold was significantly increased (p<0.05). The above data results indicate that TBF and ALA treatment can improve sensory abnormalities and neurological function in DM rats. Specific results are shown in Tables 1 and Figure 1 .

[0059] Table 1 Effects of Tangbi Recipe on thermal pain threshold and mechanical pain threshold in STZ-induced diabetic rats

[0060] Note: #compared with the control group, #p<0.05; ##p<0.01; *compared with the STZ group, *p<0.05; **p<0.01.

[0061] 3.2 Nerve conduction velocity of rats in each group Nerve conduction velocity was measured in each group of rats after 12 weeks of drug intervention. Treatment with 0.56 g / kg TBF and ALA significantly increased SNCV and MNCV (p < 0.05). Treatment with 1.12 g / kg TBF also significantly ameliorated the STZ-induced decrease in MNCV (p < 0.05). Figure 2 Results in C and D show that compared with the CTRL group, the amplitudes of sensory and motor nerve action potentials in STZ rats were significantly reduced at 12 weeks (p<0.05). However, after treatment with 1.12 g / kg of TBF and ALA, the amplitudes of motor nerve action potentials in rats were significantly increased (p<0.05), but TBF and ALA had no effect on the amplitudes of sensory nerve action potentials. Figure 2 .

[0062] 3.3 Blood perfusion around the foot pad and sciatic nerve of rats in each group LSCI results showed that the blood perfusion around the footpad and sciatic nerve of rats in the STZ group was significantly lower than that in the CTRL group (p<0.05). After 12 weeks of TBF treatment, the blood perfusion around the footpad and sciatic nerve of STZ-induced diabetic rats was significantly improved (p<0.05). These results indicate that TBF can improve peripheral vascular function and increase blood perfusion in DPN rats. Figure 3 .

[0063] 3.4 Observation of sciatic nerve histopathological morphology 3.4.1 H&E staining results of rat sciatic nerve like Figure 4 As shown, the myelinated nerve fibers in the sciatic nerves of rats in the CTRL group were arranged in an orderly manner, with normal axonal morphology. In the sciatic nerves of rats in the STZ group, loose and irregular nerve fiber arrangement was observed. The axon was located at the center of the nerve fiber, and the myelin sheath adhered to the axonal surface, forming a concentric lamellar structure. Numerous myelin sheaths and axonal swellings were observed (black arrows). Compared with the STZ group, the myelinated nerve fibers in the sciatic nerves of rats in the TBF and ALA groups were arranged more regularly, with milder pathological changes. The myelinated nerve fibers in some areas of the sciatic nerve tissue were slightly disorganized.

[0064] 3.4.2 Results of LFB staining of rat sciatic nerve like Figure 5 As shown, the myelin sheath structure of the sciatic nerves of rats in the CTRL group remained intact, with normal morphology and no degeneration or dissolution. In contrast, the myelin sheaths of the sciatic nerves of rats in the STZ group were darker in color and showed swelling, spherical curvature, and fractures. Compared with the STZ group, the myelin sheaths of the sciatic nerves of rats in the TBF and ALA groups were more regularly arranged and had less severe myelin lesions.

[0065] 3.4.3 Results of glycine silver staining of rat sciatic nerve like Figure 6 As shown, the sciatic nerve fibers of rats in the CTRL group were neatly arranged and had normal myelin sheaths. In contrast, the sciatic nerve fibers of rats in the STZ group were disorganized, with swelling, vacuolation, and degeneration of the axons. In the TBF and ALA groups, a few nerve fibers also showed swelling, vacuolation, and degeneration, but to a lesser extent than in the STZ group.

[0066] 3.4.4 Nissl staining results of rat sciatic nerve like Figure 7 As shown, the neuronal structure of rats in the CTRL group was intact, the cell morphology was normal, the cell nucleus boundaries were clear, and Nissl bodies were visible in blue. Compared with the CTRL group, the number of neurons in the STZ group decreased, the cell morphology changed, and the neuronal density per unit area decreased (p < 0.05). After 12 weeks of treatment, the neuronal morphology of the TBF and ALA groups was significantly improved compared with the STZ group, with an increase in the number of neurons and an increase in the neuronal density per unit area (p < 0.05). 3.5 Ultrastructure of sciatic nerve Transmission electron microscopy showed that TBF or ALA treatment could improve myelin demyelination and effectively correct axonal atrophy ( Figure 8A). Furthermore, compared with the CTRL group, the STZ group showed a significant decrease in the G-ratio (axon diameter / fiber diameter), a key indicator of myelin integrity (p < 0.01). Treatment with 0.56 g / kg and 1.12 g / kg of TBF and ALA significantly increased the G-ratio (p < 0.05). These results suggest that TBF can significantly rescue STZ-induced sciatic nerve axonal degeneration and myelin damage.

[0067] 4. Conclusion Tangbi prescription can reduce pain sensitivity in DM rats, increase nerve conduction velocity, improve blood perfusion around the foot pad and sciatic nerve, improve neurological phenotype, and restore neuronal function. It can also improve sciatic nerve pathological damage in DM rats.

[0068] Example 3: Animal and cell experimental mechanism studies 1 Materials and reagents 1.1 Materials 1.1.1 Experimental animals The experimental animals were the same as those in Example 2 1.1.2 Cell lines Schwann cells (SCs) were purchased from the National Cell Line Resource Infrastructure.

[0069] 1.2 Experimental drugs Preparation of TBF: As in Example 2, dissolve TBF in sterile PBS to prepare a 10 mg / mL TBF stock solution. Store at -20°C until ready for use. Dilute with culture medium to the desired concentration before use.

[0070] Glucose solution preparation: Dissolve 3.6 g glucose in 10 ml sterile PBS to prepare a 2 mol / L stock solution. Store at -20°C until ready for use. Dilute with culture medium to the desired concentration.

[0071] To prepare mannitol solution: Dissolve 3.644 g of mannitol in 10 ml of PBS to obtain a 2 mol / L stock solution. Store at -20°C until ready for use. Dilute with culture medium to the desired concentration.

[0072] Preparation of AMPK Activator (AICAR): Prepare a stock solution in DMSO to a final concentration of 0.5 mol / L. Store at -20°C until ready for use. Dilute to the desired concentration in culture medium, maintaining a final DMSO concentration of 1%. Filter sterilize.

[0073] 2 Methods 2.1 Animal grouping and medication: Same as Example 2.

[0074] 2.2 Animal sampling After 12 weeks of continuous gavage, sodium pentobarbital was injected intraperitoneally. Rats were placed in a prone position under deep anesthesia. One section of the left sciatic nerve tissue was placed in a cryovial and stored in liquid nitrogen, while the other section was fixed in 4% paraformaldehyde solution. Blood was collected from the abdominal aorta and centrifuged at 3000 rpm / min for 20 minutes at 4°C, and the supernatant was collected.

[0075] 2.3 Animal index testing 2.3.1 Determination of IL-6, TNF-a, and IL-1β levels in rat serum 2.2 serum was collected and the levels of IL-6, TNF-a and IL-1β in rat serum were detected by enzyme-linked immunosorbent assay (ELISA). The samples were appropriately diluted, and then IL-6, TNF-α, and IL-1β standard dilutions of varying concentrations were prepared for standard curve generation. 100 μL of each treated sample and standard dilution was added to a microplate pre-coated with anti-rat IL-6, TNF-α, or IL-1β. The plates were incubated at 37°C for 90 min. The solution was then aspirated, and 100 μL of biotinylated anti-rat IL-6, TNF-α, and IL-1β working solution was added to each well. The plates were incubated at 37°C for another 60 min, followed by three washes. Then, 100 μL of HRP conjugate working solution was added to each well. The plates were incubated at 37°C for another 60 min, followed by three washes. 90 μL of preheated 30°C TMB substrate solution was added, and the plates were incubated at 37°C in the dark for 20 min. TMB stop solution was added, and the absorbance was measured at 450 nm using a microplate reader. Samples were calculated by multiplying the appropriate dilution factor.

[0076] 2.3.2 Determination of SOD and MDA levels in rat serum The total SOD content in rat serum was determined by colorimetry, and the MDA expression level in rat serum was detected by thiobarbituric acid method. The operation was performed strictly according to the kit instructions.

[0077] 2.3.3 TUNEL assay for sciatic nerve apoptosis Paraffin sections were dewaxed to water, and 20 μg / ml DNase-free proteinase K was added dropwise for 15–30 min at 20–37°C. The sections were then washed three times with PBS, and 50 μL of the pre-prepared TUNEL detection solution was added. The sections were washed three times with PBS again, and finally, the sections were sealed with anti-fluorescence quenching sealing solution and photographed under a fluorescence microscope for analysis.

[0078] 2.3.4 Immunohistochemistry to detect S100β protein expression Rat sciatic nerve tissue was dehydrated, wax-impregnated, embedded, and sectioned. The sections were then washed sequentially in deparaffinizing solution, anhydrous ethanol, and distilled water. Following antigen retrieval, the sections were incubated in 3% hydrogen peroxide at room temperature in the dark for 25 minutes. Washed three times with PBS for 5 minutes each, dried, and then serum-blocked. The target primary antibody and HPR-labeled secondary antibody were added, and the sections were developed with DAB. Nuclei were counterstained. Finally, the sections were dehydrated, mounted, and photographed under a microscope for analysis.

[0079] 2.3.5 Western Blot detection of rat sciatic nerve-related protein expression Protein was extracted from rat sciatic nerve tissue and the protein concentration was determined using a BCA protein assay kit. Subsequently, SDS-polyacrylamide gel electrophoresis was performed, and the membrane was transferred to the membrane for blocking and incubation with primary antibodies, including anti-phospho-dynamin-related protein 1 (Drp1) (Ser 616, 1:1000), anti-Drp1 (1:1000), anti-mitochondrial fission protein 1 (Fis1) (1:2000), anti-mitochondrial fusion protein 1 (Mfn1) (1:5000), anti-optic atrophy protein 1 (Opa1) (1:1000), anti-mitochondrial fusion protein 2 (Mfn2) (1:1000), anti-phospho-AMPK (Thr172) (1:5000), anti-AMPK (1:1000), anti-peroxisome proliferator-activated receptor coactivator 1α (PGC-1a) (1:1000), and anti-β-tubulin (1:5000). The primary antibody was incubated overnight, and the sections were washed with TBST five times for 5 min each time, followed by incubation with the corresponding secondary antibody; the sections were washed with TBST again five times for 5 min each time, and finally the color was developed by chemiluminescence.

[0080] 2.4 Cell culture and treatment 2.4.1 Cell culture Cell recovery: Remove the cryovial containing SCs from liquid nitrogen and thaw in a 37°C water bath for 2 min. Transfer the cells to a 1.5 mL EP tube and centrifuge at 1000 rpm for 5 min. Discard the supernatant and resuspend the cells in 2 mL of culture medium. Transfer the tube to a T25 culture flask and add 6 mL of culture medium. Then, culture the tube in a 37°C, 5% CO2 cell culture incubator and change the medium every other day.

[0081] Cell passaging: When the cell confluence reaches more than 85%, discard the old culture medium, wash the cells with PBS buffer, add 1 mL of trypsin to digest for 1 min, add 2 mL of complete culture medium to terminate the digestion, pipette the flask wall to collect the cells, centrifuge at 1000 rpm for 5 min, discard the supernatant, add complete culture medium and pipette evenly, then transfer SCs to a new T25 culture flask at a ratio of 1:3, add complete culture medium to make up to 6 mL, and continue culturing in a cell culture incubator.

[0082] Cell freezing: When the cell confluence reaches more than 85%, discard the culture medium, wash the cells with PBS buffer, add 1 mL of trypsin to digest for 1 min, add complete culture medium to terminate the digestion, blow the bottle wall to collect Schwann cells, and maintain the Schwann cell density at 10 7 / mL, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 1mL of freezing solution into the cryotube, seal it, place it in a -80℃ slow freezing box, and finally store it in liquid nitrogen.

[0083] 2.4.2 Model construction and drug treatment Cell culture conditions: (1) Complete culture medium includes DMEM low-glucose medium, 1% penicillin-streptomycin, and 10% fetal bovine serum. (2) Incubator conditions: Temperature set to 37 °C, CO2 concentration to 5%. (3) Cells were passaged when they reached the logarithmic growth phase and the confluence was approximately 80%.

[0084] Cell grouping: (1) Construction of a high-glucose-damaged DPN cell model: cells were exposed to 30 mM high glucose (HG) for 24, 48, and 72 h. (2) Elimination of the effect of osmotic pressure on cell viability: cells were treated with 5.56 mM normal glucose (NG) + 24.4 mM mannitol medium for 24, 48, and 72 h. (3) Effect of TBF on the viability of HG-exposed cells: cells were treated with HG + different concentrations of TBF (50, 100, 200, 400, 800, and 1000 μg / mL) for 48 h. (4) The effects of TBF on HG-induced ROS generation, mitochondrial membrane potential (MMP), apoptosis, ATP, SOD, MDA, transmission electron microscopy and related protein expression levels were analyzed. The following groups were established: NG group: SCs were cultured in 5.6 mM glucose culture medium; HG group: SCs were cultured in high glucose (30 mM glucose) culture medium; TBF group: SCs were grown in HG culture medium treated with TBF (HG + 50, 100, 200 μg / mL TBF); AICAR group: SCs were grown in HG + 0.5 mM AICAR-treated culture medium.

[0085] 2.5 Index detection 2.5.1 Cell viability assay Cell proliferation activity was assessed using a cell counting kit-8 (CCK-8). First, SCs were seeded in a 96-well plate (100 μL / well) with 3 × 10 3 cells. Place the 96-well plate in an incubator. After incubation, add 10 μL of CCK solution to each well and incubate at 37°C for 1 hour. Finally, use a microplate reader to measure absorbance at a wavelength of 450 nm. Cell viability in the NG group was set to 1, representing the baseline level.

[0086] 2.5.2 ROS Detection ROS were quantified using a ROS detection kit. Briefly, cells were exposed to 10 μM 2',7'-dichlorofluorescein diacetate (DCFH-DA) and incubated at 37°C for 30 min, and ROS levels were quantified using flow cytometry.

[0087] 2.5.3 JC-1 test Use the JC-1 staining kit to assess changes in mitochondrial membrane potential (MMP). First, aspirate the old culture medium and wash the cells once with PBS. Then, add 1 ml of cell culture medium and 1 ml of JC-1 staining solution and mix thoroughly. Incubate the cells in a 37°C cell culture incubator for 20 minutes. After incubation, aspirate the supernatant and wash twice with JC-1 staining buffer. Finally, add 2 ml of cell culture medium and immediately examine the cells under a fluorescence microscope and photograph them for analysis.

[0088] 2.5.4 Cell apoptosis detection After trypsin digestion, collect the cells by centrifugation. Wash the cells twice with pre-chilled PBS and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and resuspend the cells in 1x Binding Buffer. Transfer 100 μL of the cell suspension to a 5 mL flow cytometer. Add 5 μL of Annexin V-FITC and 5 μL of PI, mix gently, and incubate at room temperature in the dark for 15 minutes. Add 400 μL of 1x Binding Buffer and mix gently. Finally, analyze the cells on a flow cytometer. 2.5.5 ATP Assay Aspirate the culture medium and add 200 μL of lysis buffer to each well of a 6-well plate to lyse the cells. Repeatedly pipette or shake the plates to ensure thorough cell lysis. After lysis, centrifuge at 12,000 g for 5 minutes at 4°C and remove the supernatant. Dilute the ATP standard solution with ATP assay lysis buffer to an appropriate concentration gradient for the standard curve. Subsequently, add 100 μL of the pre-prepared ATP assay working solution to each well. Incubate at room temperature for 3–5 minutes, then add 20 μL of sample or standard to each well. Mix quickly with a pipette and measure the RLU using a chemiluminescence microplate reader.

[0089] 2.5.6 MDA and SOD detection Same as 2.3.2 2.5.7 Western Blot Detection of the Expression of AMPK Pathway and Mitochondrial Dynamics-Related Proteins in SCs Same as 2.3.5 2.5.8 Transmission electron microscopy of cells The cells were collected by centrifugation, resuspended and fixed at 4°C in electron microscopy fixative for 2-4 h, pre-embedded in agar, and fixed with 1% osmium hydroxide in 0.1 M phosphate buffer PB (pH 7.4) for 2 h at room temperature in the dark. The cells were rinsed three times with 0.1 M phosphate buffer PB (pH 7.4), each for 15 min. The cells were dehydrated in ascending order with 30%-50%-70%-80%-95%-100%-100% alcohol for 20 min each, and twice with 100% acetone for 15 min each. The cells were then infiltrated, embedded, polymerized, sectioned, and stained, and observed under a transmission electron microscope for image analysis.

[0090] 3. Results 3.1 Comparison of serum IL-6, TNF-a, and IL-1β expression levels in rats of each group like Figure 9 As shown, STZ-induced DPN rats showed increased release of serum proinflammatory cytokines IL-6, TNF-a, and IL-1β ( p <0.05), while TBF treatment can significantly reverse the abnormal changes of these inflammatory factors. In addition, ALA treatment also has an inhibitory effect on the increase in the expression level of IL-6 inflammatory factors ( p <0.05), but although there was a certain degree of inhibition on the changes of TNF-a and IL-1β, the difference was not statistically significant. The results showed that TBF can effectively inhibit the inflammatory response in DM rats.

[0091] 3.2 Comparison of serum SOD and MDA expression levels in rats of each group like Figure 10 As shown in Figure 2, compared with the CTRL group, the expression level of serum SOD in the STZ group was decreased, while the expression level of MDA was increased (p <0.05). Compared with the STZ group, the serum SOD levels of rats in the 0.28 g / kg TBF group and ALA group were significantly increased ( p <0.05); meanwhile, the serum MDA levels of rats in the 1.12 g / kg TBF group and the ALA group were significantly decreased ( p <0.05). The results showed that TBF could inhibit the generation of oxygen free radicals and the increase of peroxidation level in STZ-induced DM rats, and protect the activity of SOD.

[0092] 3.3 Results of TUNEL detection of rat sciatic nerve apoptosis like Figure 11 As shown, the nuclei of SCs in the CTRL group were stained with DAPI and then stained blue under UV excitation, while the nuclei of apoptotic cells were stained green. No apoptotic cells were observed in the CTRL group. Compared with the CTRL group, the green staining was enhanced in the STZ group. Compared with the STZ group, apoptosis was reduced in the TBF and ALA groups.

[0093] Effect of TBF on S-100β protein expression in rat sciatic nerve Immunohistochemistry was used to detect the changes in the expression levels of S-100β protein in each group. Figure 12 As shown, compared with the CTRL group, the S-100β protein positive area ratio in the STZ group was significantly reduced ( p <0.01), after 12 weeks of TBF and ALA intervention, the positive area ratio of S-100β protein increased, and the difference was statistically significant ( p <0.05).

[0094] Effects of TBF on the expression of AMPK pathway and mitochondrial dynamics-related proteins in rat sciatic nerve Based on the potential mechanisms predicted by previous network pharmacology studies and the current research status, we investigated the effects of TBF on the AMPK signaling pathway and downstream mitochondrial dynamics. Figure 13 As shown, compared with the CTRL group, the ratio of p-AMPK to AMPK and the expression of PGC-1α protein level in the STZ group were decreased ( p <0.05), indicating that AMPK pathway activation was inhibited. However, this inhibition was reversed after treatment with TBF and ALA ( p<0.05). Notably, TBF attenuated the elevated levels of mitochondrial fission-related proteins in the sciatic nerves of STZ-induced diabetic rats. The ratio of p-Drp1 to Drp1 and Fis1 levels were significantly decreased in the TBF and ALA groups. Furthermore, the expression of mitochondrial fusion-related proteins was increased. Compared with the STZ group, the levels of Mfn1, Mfn2, and Opa1 were significantly increased in the TBF and ALA groups. These results suggest that TBF can activate the AMPK signaling pathway to regulate mitochondrial homeostasis.

[0095] 3.6 Establishment of HG-induced SCs injury model SCs are considered to be one of the possible causes of repairing nerve damage, and HG is considered to be the most important feature of DM. The American Diabetes Association defines the average fasting blood glucose level as less than 5.6 mM. In order to simulate the uncontrolled DM state, we used 30 mM glucose medium to detect the effect of HG on SCs viability. In addition, we also used 5.6 mM glucose medium as a reference for normal physiological levels. SCs were cultured under HG conditions for 24, 48, and 72 h, and the cytotoxic effect of HG was evaluated using the CCK-8 method. Taking into account the osmotic effect caused by HG, we used a combination of 5.6 mM NG and 24.4 mM mannitol as an osmotic pressure control. The results showed that compared with the NG group, the cell viability was significantly reduced after 48 and 72 h of culture under HG conditions ( p <0.05), however, no significant effect was observed within 24 h. No significant difference was found between the NG + mannitol group and the NG group, so the effect of osmotic pressure can be excluded. Figure 14 .

[0096] Effect of TBF on the viability of HG-induced SCs like Figure 15 As shown in Figure 2, under HG conditions, treatment with TBF at concentrations of 50, 100, 200, 400, and 800 μg / mL for 48 h increased cell viability. This suggests that TBF can reverse the HG-induced decrease in cell viability. Notably, TBF at a concentration of 1000 μg / mL was toxic to HG-treated cells. Based on this, we selected 50, 100, and 200 μg / mL TBF concentrations for our study.

[0097] Effect of TBF on ROS production in HG-exposed SCs During the hyperglycemic response of DM, ROS are generated and have toxic effects on cells. To investigate the potential protective effect of TBF against HG-induced oxidative toxicity, we used DCFH2-DA staining to assess ROS levels. Flow cytometry analysis showed that ROS accumulation in SCs was significantly enhanced after 48 h of HG treatment compared with the NG group (p <0.01). However, after treatment with 50, 100, and 200 μg / mL of TBF and AICAR, this increase was significantly inhibited ( p <0.05). The results showed that TBF can alleviate oxidative stress under HG stimulation. Figure 16 .

[0098] Effects of TBF on mitochondrial membrane potential (MMP) in HG-treated SCs MMP is often regarded as one of the important indicators for evaluating mitochondrial function. Increased ROS production will further damage mitochondria, leading to a decrease in MMP. Figure 17 As shown, JC-1 staining method showed that the ratio of red fluorescence (aggregates) to green fluorescence (monomers) in the HG group was lower than that in the NG group ( p <0.05), indicating that MMP decreased and HG caused mitochondrial damage. However, pretreatment with 50 μg / mL and 200 μg / mL TBF and AICAR partially reversed this trend and stabilized MMP ( p <0.05). These results suggest that TBF has a protective effect in protecting SCs from HG-induced mitochondrial dysfunction.

[0099] 3.10 Effect of TBF on apoptosis of HG-treated SCs Apoptosis of SCs is crucial in the pathogenesis of DPN. Therefore, we next investigated whether TBF could reduce cell apoptosis under HG conditions. Figure 18 As shown in Figure 2, after 48 h of HG treatment, the level of cell apoptosis increased significantly ( p <0.01). However, TBF and AICAR pretreatment reduced the level of cell apoptosis ( p <0.05).

[0100] Effect of TBF on ATP production in SCs exposed to HG like Figure 19 As shown in Figure 3, SCs ATP production was significantly reduced under HG exposure, and AICAR significantly enhanced the inhibition of HG on ATP ( p <0.01). Notably, TBF significantly increased ATP production in SCs in a dose-dependent manner.

[0101] 3.12 Effects of TBF on MDA and SOD in SCs after HG exposure like Figure 20 As shown in Figure 2, after HG treatment, the SOD expression level of SCs was significantly decreased ( p <0.05, while MDA levels were significantly increased ( p<0.05). TBF at a concentration of 100 μg / mL could significantly reverse the decrease in SOD levels stimulated by HG ( p <0.05), and 200 μg / mL TBF and AICAP could reduce the expression of MDA ( p <0.05). The results indicate that TBF can protect SCs from the damage of superoxide free radicals and oxidative stress induced by HG.

[0102] Effects of TBF on the expression of proteins related to the AMPK pathway and mitochondrial dynamics in SCs induced by HG exist Figure 21 The results showed that exposure of SCs to HG conditions resulted in a significant decrease in the p-AMPK / AMPK ratio and PGC-1a levels. Compared with the HG group, the TBF group significantly increased the p-AMPK / AMPK ratio and PGC-1a expression levels in SCs, and long-term AIACR treatment reversed this trend. These results indicate that TBF can activate the AMPK pathway in SCs induced by HG, which is consistent with the results of animal experiments. In addition, we also observed that the degree of activation of the AMPK pathway by TBF was comparable to that of AICAR. Subsequently, we found that the expression levels of Mfn1, Mfn2, and Opa1 proteins in SCs exposed to HG conditions were significantly downregulated. In addition, the p-Drp1 / Drp1 ratio and the expression level of FIS1 were significantly increased in the HG group compared with the NG group. Compared with the HG group, the p-Drp1 / Drp1 ratio and FIS1 protein expression level were significantly decreased in the TBF and AICAR groups, while the expression levels of Mfn1, Mfn2 and Opa1 proteins were upregulated.

[0103] 3.14 Transmission electron microscopy observation of the effects of TBF on the ultrastructure of mitochondria in HG-treated SCs like Figure 22 As shown in the figure, compared with the NG group, the number of mitochondria in SCs in the HG group was larger, and their morphology was rounder, smaller, and more fragmented. Some mitochondria were significantly swollen, with local matrix dissolution and cristae broken or disappeared. After TBF or AICAR treatment, the mitochondrial morphology was better than that in the HG group.

[0104] 4. Conclusion TBF promoted mitochondrial fusion and inhibited mitochondrial fission by activating the AMPK signaling pathway, improved STZ-induced apoptosis of sciatic nerves in DM rats and SCs oxidative stress and apoptosis under HG exposure, and alleviated mitochondrial structural and functional damage, thereby exerting a positive neuroprotective effect.

[0105] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A traditional Chinese medicine composition for preventing and / or treating diabetic peripheral neuropathy, comprising the following raw materials in parts by weight: 6-40 parts of angelica sinensis, 9-120 parts of astragalus, 0.5-15 parts of notoginseng, 3-30 parts of sappan wood, 3-30 parts of cassia twig, 3-100 parts of ligusticum chuanxiong, 5-120 parts of white peony root, 3-200 parts of heishunpian, and 9-70 parts of mulberry branch.

2. The Chinese medicine composition according to claim 1, wherein: The Chinese medicine composition is prepared from the following raw materials in parts by weight: 6-20 parts of angelica sinensis, 9-30 parts of astragalus, 0.5-9 parts of notoginseng, 3-27 parts of sappan wood, 3-27 parts of cassia twig, 3-30 parts of chuanxiong, 6-20 parts of white peony root, 3-20 parts of heishunpian, and 9-30 parts of mulberry branch.

3. The Chinese medicine composition according to claim 1, wherein: The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 9 parts of angelica sinensis, 15 parts of astragalus, 3 parts of notoginseng, 9 parts of sappan wood, 9 parts of cinnamon twig, 15 parts of ligusticum chuanxiong, 9 parts of white peony root, 3 parts of heishunpian, and 15 parts of mulberry branch.

4. The method for preparing the Chinese medicine composition according to any one of claims 1 to 3, comprising the steps of: mixing the raw materials, decocting with water for extraction, and collecting the supernatant after the extraction is completed.

5. The method for preparing the Chinese medicine composition according to claim 4, wherein: After mixing the raw materials, soak them in cold water for 30 minutes, adding water that exceeds the medicine surface by 2-3 cm. Boil the first decoction over high heat (strong fire), then keep boiling over low heat (gentle fire) for 25-35 minutes, until the medicine liquid is 30-50% of the initial water amount, pour it out, add water again for the second decoction, just enough to soak the medicine surface, boil over high heat, then keep boiling over low heat (gentle fire) for 25-35 minutes, until the medicine liquid is 30-50% of the initial water amount, pour it out, and mix the two decoctions to obtain the product.

6. The method for preparing the Chinese medicine composition according to claim 5, wherein: The method further comprises the step of freeze-drying the obtained supernatant to obtain freeze-dried powder.

7. A drug for preventing and / or treating diabetic peripheral neuropathy, the active ingredient of which comprises the traditional Chinese medicine composition according to any one of claims 1 to 3 or the traditional Chinese medicine composition prepared by the method according to any one of claims 4 to 6.

8. The drug according to claim 7, characterized in that: The dosage form of the Chinese medicine composition is an oral dosage form; further, the oral dosage form is selected from any one of the following: granules, capsules, tablets, oral liquids, powders, pills, dripping pills, sustained-release preparations, oral liquids, mixtures and syrups.

9. The Chinese medicine composition according to any one of claims 1 to 3 or the medicine according to claim 7 or 8, characterized in that: The pharmaceutical composition or medicine is a medicine suitable for treating pain, numbness and paresthesia symptoms caused by diabetic peripheral neuropathy.

10. The Chinese medicine composition according to any one of claims 1 to 3 or the medicine according to claim 7 or 8, characterized in that: The prevention and / or treatment of diabetic peripheral neuropathy is embodied in at least one of the following aspects: 1) Reduce pain sensitivity; 2) Increase nerve conduction velocity; 3) Improve the decreased blood perfusion around the foot pad and sciatic nerve; 4) Improve neurological phenotype and restore neuronal function; 5) Can improve sciatic nerve pathological damage; 6) Inhibit inflammatory response; 7) Promoted mitochondrial fusion and inhibited mitochondrial fission by activating the AMPK signaling pathway; 8) Improved STZ-induced sciatic nerve apoptosis in diabetic rats; 9) Improved oxidative stress and apoptosis of Schwann cells exposed to high glucose, and alleviated mitochondrial structural and functional damage.

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