[Baimai Ointment relieves chronic pain induced by chronic compression of dorsal root ganglion in rats by regulating neuroactive ligand-receptor interaction and HIF-1 signaling pathway].

The Baimai Ointment with the effect of relaxing sinew and activating collaterals demonstrates a definite effect on Baimai disease with pain, spasm, stiffness and other symptoms, while the pharmacodynamic characteristics and mechanism of this agent remain unclear. In this study, a rat model of chronic compression of L4 dorsal root ganglion(CCD) was established by lumbar disc herniation, and the efficacy and mechanism of Baimai Ointment in the treatment of CCD were preliminarily explored by behavioral tests, side effect evaluation, network analysis, antagonist and molecular biology verification. The pharmacodynamic experiment indicated that Baimai Ointment significantly improved the pain thresholds(mechanical pain, thermal pain, and cold pain) and gait behavior of CCD model rats without causing tolerance or obvious toxic and side effects. Baimai Ointment inhibited the second-phase nociceptive response of mice in the formalin test, increased the hot plate threshold of normal mice, and down-regulated the expression of inflammatory cytokines in the spinal cord. Network analysis showed that Baimai Ointment had synergistic effect in the treatment of CCD and was related to descending inhibition/facilitation system and neuroinflammation. Furthermore, behavioral tests, Western blot, and immunofluorescence assay revealed that the pain-relieving effect of Baimai Ointment on CCD may be related to the regulation of the interaction between neuroactive ligand and receptors(neuroligands) such as CHRNA7, ADRA2A, and ADRB2, and the down-regulation of the expression of NOS2/pERK/PI3K, the core regulatory element of HIF-1 signaling pathway in spinal microglia. The findings preliminarily reveal the mechanism of relaxing sinew and activating collaterals of Baimai Ointment in the treatment of Baimai disease, providing a reference for the rational drug use and further research of this agent.

[Expert opinion on clinical application of Baimai Ointment].

Baimai Ointment, derived from Tibetan classics, is indicated for Baimai diseases(nervous system diseases) with the effects of relaxing tendons and activating collaterals. It is included in the National Basic Medical Insurance Drugs Catalogue and is widely used for neurological rehabilitation and musculoskeletal system diseases, showing efficacy and safety. Nevertheless, the description of indications, usage, and dosage in the instructions of Baimai Ointment is unclear. There has been no standard for the application of Baimai Ointment. To give full play to its effect and to guide and standardize the clinical medication, this research group organized 23 experts specializing in Tibetan medicine, traditional Chinese medicine, western medicine, evidence-based medicine, methodology, pharmacy, and bibliography from 14 organizations jointly developed this Expert opinion on clinical application of Baimai Ointment. Due to the lack of clinical evidence, this expert consensus is innovatively combined with massive ancient classics, abundant experts' experience, and comprehensive clinical evidence. After discussion and in-depth interview, the consensus on the indications, usage, dosage, duration, frequency, course, and combination protocol of Baimai Ointment has been achieved. Safety and contraindications have been clearly stated. This expert opinion can provide guidance and references for clinicians at all levels to use Baimai Ointment in a reasonable and standardized way.

[Connotation of Baimai disease and analysis of compatibility and usage of Baimai Ointment: based on Tibetan medicine theory].

Baimai is a complex of structure and function with the characteristics of wide distribution, complex structure, and multi-dimensional functions. Baimai, consisting of the channels in brain, the internal hidden channels connecting the viscera, and the external channels linking the limbs, governs the sensory, motor, and information transmission functions of human. According to Tibetan medicine, Baimai functions via "Long"(Qi) which moves in Baimai. "Long" is rough, light, cold, tiny, hard, and dynamic. The dysfunction of Baimai is manifested as numbness, swelling and pain, stiffness, atrophy, contracture, disability, hyperactivity, etc. The clinical manifestations of Baimai disease are facial paralysis, limb numbness, hemiplegia, contracture and rigidity, pain, opistho-tonos, paralysis, unconsciousness, head tremor, aphasia and tongue stiffness, and other abnormalities in facial consciousness, limb movement, and tactile sensation. Baimai Ointment for external use is used for the treatment of Baimai disease. It is mainly composed of medicinals which are spicy and bitter, warm, soft, mild, heavy, moist, and stable, and thus it is effective for the rough, light, cold, tiny, hard, and dynamic "Long" of Baimai disease. In clinical practice, it is mainly used for musculoskeletal diseases, such as osteoarthritis, scapulohumeral periarthritis, cervical spondylosis, low back pain, myofascitis, and tenosynovitis, nervous system diseases, such as paralysis and shoulder-hand syndrome, and limb stiffness caused by stroke, spastic cerebral palsy, trigeminal neuralgia, and facial neuritis, and limb motor and sensory dysfunction caused by trauma. According to the main symptoms of Baimai disease such as stiffness, rigidity, contraction, numbness, sensory disturbance and pain, clinicians should apply the Baimai Ointment via the inunction treatment of Tibetan medicine and in combination with Huo'ermai therapy and physiotherapy.

[Enhancing effects of chemical permeation enhancer propylene glycol and ultrasonic technology on transdermal permeation of Baimai Ointment].

This study aimed to improve the transdermal permeation quantity of Baimai Ointment by investigating the enhancing effects of physical and chemical permeation promoting methods on transdermal permeation of Baimai Ointment. The improved Franz diffusion cell method was used for in vitro transdermal experiment. The abdominal skin of mice was used, and the skin was treated with 3% propylene glycol in the chemical enhancement group. Ultrasonic technology was introduced in the physical enhancement group. The conditions of ultrasonic technology were optimized by single factor trial. Taking Q_(EF) and ER as the indexes of penetration promotion performance, the enhancing effects of the two methods were compared. The results showed that the promotion performance of 3% propylene glycol for ammonium glycyrrhizinate, nardosinone and curcumin of the chemical enhancement group were 1.74, 1.60, and 3.73 times higher than those of the blank group, respectively. The overall permeation efficiency of the Baimai Ointment was significantly improved. The comprehensive promoting effect on each component was curcumin>ammonium glycyrrhizinate>nardosinone. In the physical enhancement group, the penetration promoting effect of ultrasonic power 1.0 W was better than that of 2.0 W and 0.5 W, ultrasonic time 5 min was better than 3 min and 8 min, and the ultrasonic frequency 1 MHz was better than 3 MHz. Therefore, the optimal ultrasonic condition was 1.0 W-5 min-1 MHz. Under this condition, in terms of the transdermal permeation for ammonium glycyrrhizinate, the Q_(EF) and ER of the ultrasonic technology were better than those of 3% propylene glycol. In terms of the transdermal permeation for nardosinone and curcumin, the QEF and ER of 3% propylene glycol were better than those of the ultrasonic technology. Therefore, 3% propylene glycol combined with ultrasonic technology can be used to promote permeation of Baimai Ointment that contains both water-soluble and fat-soluble components in the clinical application. This study provides a theoretical basis for the clinical application of Baimai Ointment and other transdermal preparations.

[Therapeutic effect of Baimai Ointment on peripheral sensitization of chronic inflammatory pain in mice].

Chronic inflammatory pain is mainly manifested by peripheral sensitization. Baimai Ointment(BMO), a classical Tibetan medicine for external use, has good clinical efficacy in the treatment of chronic inflammatory pain, while its pharmacodynamics and mechanism for relieving peripheral sensitization remain unclear. This study established an animal model of chronic inflammatory pain induced by complete Freund's adjuvant to explore the mechanism of BMO in the treatment of chronic inflammatory pain by behavioral test, side effect assessment, network analysis, and experimental verification. The pharmacodynamics experiment showed that BMO increased the thresholds of mechanical pain sensitivity and thermal radiation pain sensitivity of chronic inflammatory pain mice in a dose-dependent manner, and had inhibitory effect on foot swelling, inflammatory mediator, and the expression of transient receptor potential vanilloid-1(TRPV1) and transient receptor potential A1(TRPA1). The results of body weight monitoring, pain sensitivity threshold detection in normal mice, rotarod performance test, and forced swimming test showed that BMO had no obvious toxic or side effect. The network analysis of 51 candidate active molecules selected according to the efficacy of BMO, content of main components, and ADME parameters showed that the inhibitory effect of BMO on chronic inflammatory pain was associated with the core regulatory elements of tumor necrosis factor(TNF) and T cell receptor signaling pathways. BMO down-regulated the protein levels of mitogen-activated protein kinase 14(MAPK14), MAPK1, and prostaglandin-endoperoxide synthase 2(PTGS2), and up-regulated the phosphorylation le-vel of glycogen synthase kinase 3 beta(GSK3 B) in the plantar tissue of mice. In conclusion, BMO can effectively relieve peripheral sensitization of chronic inflammatory pain without inducing tolerance and obvious toxic and side effects. The relevant mechanism may be related to the regulation of BMO on core regulatory elements of TNF and T cell receptor signaling pathways in surrounding tissues.

[In vitro transdermal permeation of main compositions in Baimai Ointment].

To establish a determination method for the contents of ammonium glycyrrhetate,nardosinone,and curcumin in transdermal receptor liquid of Baimai Ointment,and investigate the percutaneous permeability of Baimai Ointment and the effects of two kinds of penetration enhancers on percutaneous absorption of three components. The contents of ammonium glycyrrhetate,nardosinone,and curcumin in transdermal receptor liquid were determined by high pressure liquid chromatography( HPLC). The vertical modified Franz diffusion cell was used to perform a transdermal experiment in vitro with the abdominal skin of mice( treated and untreated). The transdermal receptor liquid was preferably used to investigate the transdermal absorption rule of the Baimai Ointment and the effect of the penetration enhancer. The results showed that the comprehensive solubility of PEG-ET-NS( 3 ∶3 ∶4) was best among three types of receptor liquid PG-ET-NS( 3 ∶3 ∶4),PEG-ET-NS( 3 ∶3 ∶4),ET-NS( 3 ∶7). PEG-ET-NS was used as the receptor liquid for in vitro transdermal experiments. The cumulative permeation area of ammonium glycyrrhetate,nardosinone and curcumin within 24 h was 5. 73,18. 99,0. 38 µg·cm~(-2)respectively. Taking QEFand ER as comprehensive evaluation indicators of permeation performance,the comprehensive penetration-promoting performance of ammonium glycyrrhizinate: 3% PEG 400-ethanol-normal saline ≈ 1. 19 times( 3%azone) = 1. 94 times( blank); comprehensive penetration-promoting performance of nardosinone: 3% PEG 400-ethanol-normal saline≈1. 28 times( 3% azone) = 1. 37 times( blank); the comprehensive penetration performance of curcumin: 3% PEG 400-ethanol-normal saline≈1. 77 times( 3% azone) ≈3. 42 times( blank). The comprehensive penetration enhancement properties of the two penetration enhancers were as follows: 3% PEG 400-ethanol-normal saline>3%azone>blank. The transdermal absorption curve of ammonium glycyrrhetate,nardosinone and curcumin in Baimai Ointment were consistent with the zero-order equation,indicating that the transdermal absorption process was irrelevant to the concentration of three components,and its was a diffusion process. This experiment provides reference for the study of ointment transdermal preparations.

Ruyi Zhenbao Tablet and Baimai Ointment Therapy in Acute Ischemic Stroke: A Study Protocol for a Multi-Center, Randomized, Double-blinded, and Parallel-Controlled Trial.

Introduction: Stroke is characterized by high incidence, recurrence rate, and mortality. Patients with acute ischemic stroke (AIS) who are ineligible for acute revascularization therapy require more effective medication treatments. A previous clinical study showed that Ruyi Zhenbao tablets and Baimai ointments might be effective against AIS; however, high-quality clinical evidence supporting their application in AIS is lacking. To explore the efficacy of the two classic Tibetan medicines in the treatment of AIS, a randomized clinical trial will be conducted in patients with AIS who are not eligible for thrombolytic treatment. Methods: A prospective, randomized, multiple-center, double-blinded, placebo-controlled, and parallel-group trial will be conducted. We shall randomize 480 eligible participants to either the intervention or the control group. The distribution ratio of each group will be 1:1:1:1, including 120 patients each in the dual-medication group, the Baimai ointment group, the Ruyi Zhenbao tablet group, and the placebo group. Participants will be treated with medication for 8 weeks, and they will receive three follow-up visits: at 4 weeks (D29), 8 weeks (D56), and 90 days (D90) after commencing treatment. The primary outcome will be D90 change in the simplified Fugl-Meyer score from baseline to posttreatment. The secondary outcomes are as follows: D29 change of simplified Fugl-Meyer score from baseline to posttreatment; proportion of participants whose D29 NIHSS scores decreased by four or more points from baseline D90 proportion of subjects with mRS score of 0-2 (inclusive); D90 proportion of subjects with Barthel index score ≥95; D90 incidence of cardiovascular and cerebrovascular events. Safety endpoint includes mortality within 90 days; proportion of subjects with adverse events/serious adverse events within 90 days. Conclusion: This research protocol lays a solid groundwork for its practical execution. This study is poised to serve as a reference for other Tibetan medicine researchers, contributing to the reduction of stroke-related expenditures globally and, in turn, benefiting a broader population of stroke patients.

Analgesic and neuroprotective effects of Baimai Ointment on diabetic peripheral neuropathy.

ETHNOPHARMACOLOGY RELEVANCE: Baimai (BM) ointment, a traditional Tibetan medicine, has been widely used to treat "white vein" disease, paralysis, hemiplegia and claudication caused by trauma, because of its great effects on muscle stretching and collateral activation. As one of the most terrible complications in diabetes patients, diabetes peripheral neuropathy (DPN) is mainly manifested as abnormal pain or numbness in extremities. However, whether BM ointment is a potential drug for DPN treatment is unclear. AIMS OF THE STUDY: The aim of this study was to investigate the therapeutic effects of BM on DPN in a high-fat diet/low-dose of streptozotocin induced type 2 diabetes rat model and explore underlying mechanisms. METHODS: The chemical components of BM were determined by high performance liquid chromatography (HPLC), and the possible targets and related pathways candidates involved in the effects of BM on DPN were predicted using network pharmacology methods. Next, the effects of different doses (1.5, 3.0 and 6.0 g/kg) of BM on physiological changes, pain behaviors, motor nerve conduction velocity (MNCV) in DPN rats were assessed and compared with placebo- and mecobalamine (Meco)-treated DPN controls. Then, the effects of BM on the expression of pain associated genes as well as the phosphorylation of PI3K/AKT and MAPKs pathways in DRG of DPN rats were examined. RESULTS: Through HPLC analysis, curcumin was identified as one of the primary contents of BM. The information from network pharmacology indicated a series of target candidates for BM including IL6, IL10, TNF, CCL2, CXCL12, EGF, VEGFA, BDNF, TGFß1 and TNF, as well as PI3K-AKT and MAPK signaling pathways. Topical treatment of BM significantly improved the hypersensitivity of mechanical and thermal pain, MNCV and the morphological changes and demyelination of sciatic nerve fibers, without affecting the body weight, serum metabolism or blood glucose. The up-regulated levels of neuropeptides Cgrp, Sst, Sp and chemokines Ccl2 and Ccl3 along with the abnormal expression of p-P38, p-ERK and p-AKT in the DRG of DPN rats were alleviated by BM application. CONCLUSION: BM ointment has great activities in relieving pain hypersensitivity, neuroprotecting peripheral nerves damage caused by DPN, which may be related to the inhibition of related neuropeptide (Cgrp, Sst, Sp) and chemokine (Ccl2, Ccl3) expression and the regulation of PI3K/AKT and MAPKs signaling pathways in DRG.

Study on the Mechanism of Baimai Ointment in the Treatment of Osteoarthritis Based on Network Pharmacology and Molecular Docking with Experimental Verification.

Purpose: The external preparation of the Tibetan medicine formula, Baimai ointment (BMO), has great therapeutic effects on osteoarthritis (OA). However, its molecular mechanism remains almost elusive. Here, a comprehensive strategy combining network pharmacology and molecular docking with pharmacological experiments was adopted to reveal the molecular mechanism of BMO against OA. Methods: The traditional Chinese medicine for systems pharmacology (TCMSP) database and analysis platform, traditional Chinese medicine integrated database (TCMID), GeneCards database, and DisGeNET database were used to screen the active components and targets of BMO in treating OA. A component-target (C-T) network was built with the help of Cytoscape, and the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment through STRING. Autodock Tools which was used to dock the key components and key target proteins was analyzed. Animal experiments were performed to verify the key targets of BMO. Hematoxylin-eosin and toluidine blue staining were used to observe the pathology of joints. Protein expression was determined using enzyme-linked immunosorbent assay. Results: Bioactive compounds and targets of BMO and OA were screened. The network analysis revealed that 17-ß-estradiol, curcumin, licochalone A, quercetin, and glycyrrhizic acid were the candidate key components, and IL6, tumor necrosis factor (TNF), MAPK1, VEGFA, CXCL8, and IL1B were the candidate key targets in treating OA. The KEGG indicated that the TNF signaling pathway, NF-κB signaling pathway, and HIF-1 signaling pathway were the potential pathways. Molecular docking implied a strong combination between key components and key targets. The pathology and animal experiments showed BMO had great effects on OA via regulating IL6, TNF, MAPK1, VEGFA, CXCL8, and IL1B targets. These findings were consistent with the results obtained from the network pharmacology approach. Conclusion: This study preliminarily illustrated the candidate key components, key targets, and potential pathways of BMO against OA. It also provided a promising method to study the Tibetan medicine formula or external preparations.