Professor Linlan’s Clinical Experience In The Treatment Of DPN Data Mining And The Related Mechanism Research

Diabetic peripheral neuropathy (DPN) is one of the most common chronic complications of diabetes. The disease can be serious impact on the patient’s survival quality, increase diabetes patient’s disability and mortality rates. At present the pathogenesis of this disease is still not clear, effective drugs for prevention and treatment of this disease is very limited. Professor LinLan studys prevention and treatment of diabetes and its complications for over50years, she has very understanding of TCM etiology, pathology and treatment of DPN, and creates a number of effective method of treatments. TangTongfang is one of her essence of clinical prevention and treatment of DPN. This paper guided by the academic thought of Professor Lin-dialectical theory of three types of diabetes. First we excavation and finishing her clinical experience in the treatment of DPN, then using cultured Schwann cells for the study, using serum pharmacology method, from apoptosis signaling pathways to explore the possible molecular mechanism of TangTongfang.1. Clinical studyObjective:To mine and analyze professor Lin’s clinical cases of DPN.To conclude the understanding from Professor Lin of the etiology and pathogenesis of diabetic peripheral neuropathy (DPN) in the perspective of traditional Chinese medicine, the syndrome differentiation of this lesion, and the application characteristics of prescription. To provide clinical evidence basis for concluding, refining and optimizing the principles of the prevention and therapy of DPN with traditional Chinese medicine, and to enhance the study efficiency and level with the experience from Professor Lin.Methods:The clinical history of107patients diagnosed with DPN in the endocrinology clinic of Guang’anmen Hospital with470clinic visits from January2011to January2014were collected and structuralized into the electronic medical record of clinical treatment. All data of electronic medical record, rating scale, assay result and prescription were imported into the data warehouse, and were analyzed with multi-dimensional searching and analyzing platform. Statistical analysis was performed on such items as the general information of DPN patients, result of laboratory examination, characteristics of symptoms and syndrome types, medication feature of Professor Lin, and evaluation of clinical efficacy.Results:1. Statistics of general information from DPN patients:Among the105cases,52.4%were male and47.6%were female;77.1%were with family history and22.9%were not;14.3%were younger than50,32.4%aging from50to59,28.6%aging from60to69, and24.8%were older than70;3.8%suffered from diabetes with the course of disease less than1year,59.2%with the disease course from1to10years,30%with the disease course from11to20years, and6.7%with the disease course longer than20years.2. Patient symptom analysis:According to the statistical results of the frequency of each symptom of patients, limb numbness was most occurred in the symptoms of sensory and motor function impairment, and then were limb coldness, limb pain and limb edema in the next place, while limb rigidity and paralysis were the least noted symptoms. Based on the frequency of other symptoms and tongue vein syndrome, symptoms of blood stasis and qi deficiency were most noted, then the symptoms of yin and yang deficiencies were of medium frequency, while excessive heat syndrome was least noted.3. Analysis of syndrome factors:In single syndrome analysis of all syndromes of DPN, the most common ones were qi deficiency syndrome, yang deficiency syndrome and yin deficiency syndrome while the frequencies of blood stasis syndrome, phlegm dampness syndrome and excessive heat syndrome were in the second place. However, the combination of syndromes was common. According to the combinations of two syndromes, three syndromes and four syndromes, the deficiency of both qi and yin showed the highest occurrence rate. The combination of blood stasis with qi and yin deficiency showed higher occurrence rate than that of the combination of phlegm dampness with qi and yin deficiency. Yin and yang deficiency showed the second highest occurrence rate, and the frequency of the combination of phlegm dampness with yin and yang deficiency was higher than that of the combination of blood stasis with yin and yang deficiency. Deficiency of yin and excessive heat syndrome showed the lowest occurrence rate, and the frequencies of the combinations of phlegm dampness and blood stasis with deficiency of yin and excessive heat syndrome were all less than10times. Based on data mining results, it could be concluded that the major syndrome types of DPN were the combination of blood stasis with qi and yin deficiency and the combination of phlegm with yin and yang deficiency.4. Analysis of clinical medication adopted by Professor Lin:All prescriptions of traditional Chinese medicine prescribed by LinLan were collected from the treatment of105DPN patients and in470clinic visits. All herbal medicines in the prescriptions were ranked according to their overall frequencies, and69herbal medicines were defined as high frequency herbal medicine as they were prescribed with more than10times. The69medicines were classified according to efficacy with references of Traditional Chinese Pharmacology7th edition and Chinese MateriaMedica. The results demonstrated that the herbal drugs most adopted by Professor Lin in treating DPN were deficiency-nourishing drugs and drugs to promote blood circulation and remove blood stasis, and the secondly adopted drugs were mind-easing drugs, astringent therapy drugs, qi regulating drugs, expectorant drugs, damp dispersing drugs, drugs for calming Liver to stop endogenous Wind, exterior-releasing drugs and heat-clearing drugs. Drugs for nourishing blood and drugs for nourishing qi played the major role in the application of deficiency-nourishing drugs, and drugs for yin and yang nourishment came in the second place. The69high frequency herbal medicines were also classified according to channel tropism. Herbal medicines of the same type may have multiple channel tropisms, and each channel tropism of every herbal medicine was collected. The results demonstrated that heart tropism drugs were most frequently used by Professor Lin Lan in treating DPN, then came such drugs of liver tropism, kidney tropism, lung tropism, spleen tropism, stomach tropism, large intestine tropism, gallbladder tropism, bladder tropism, pericardium tropism, small intestine tropism and triple energizer tropism.5. Clustering analysis of the high frequency herbal medicines was performed with the data mining software Liquorice based on efficacy. Statistics showed the principles of compatibility of medicines used by Professor Lin in treating DPN, which indicated the principles of the compatibility of core compounds and the principles of the compatibility of commonly used medicines.Conclusion:The theory of three syndromes differentiation of diabetes is the core theoretical basis for the clinical syndrome differentiation and therapy of diabetes. Syndrome differentiation and treatment of DPN is the extended application guided by the theory above. The morbidity basis of DPN is the deficiency of qi and yin and the deficiency of yin and yang leads to exacerbation. Blood stasis and phlegm-damp are the main pathogenic factors. The lesion of this disease involves the five-organ system with complicated symptoms. DPN can be classified into four types based on the characteristics of clinical symptoms as the deficiency of qi and yin with vessel stagnation, the deficiency of yin and yang with cold-dampness syndrome, the deficiency of liver-yin and kidney-yin with escape of blood from vessels, and the deficiency of spleen and stomach with phlegm and blood stasis syndrome. In this study, we collected the data of clinical cases in a comprehensive and standard way with the first application of the data collecting system of structuralized clinical treatment information, and analyzed the clinical experience of Professor Lin Lan in treating DPN from such perspectives as disease, symptom, syndrome, treatment, prescription and medication. Then we concluded the understanding from Professor Lin Lan of the etiology and pathogenesis of diabetic peripheral neuropathy (DPN) in the perspective of traditional Chinese medicine, the syndrome differentiation of this lesion, and the application characteristics of prescription. This study will provide some reference for the inheritanceand promotion of the experience from distinguished veteran doctors of traditional Chinese medicine.2. Experimental studyObjective:To explore the molecular mechanism of Tangtongfang in treating diabetic peripheral neuropathy from the perspective of cellular apoptosis signaling pathway.Methods:Schwann cells were incubated from the sciatic nerve of SD suckling mouse and interfered with the culture solution containing50mM high glucose to induce the apoptosis model of Schwann cell. We adopted cck-8method to screen out the optimum concentrations of drug-containing serum of Tangtongfang, Ligustrazine hydrochloride, and Astragalus polysaccharide in promoting Schwann cell proliferation, and use these drugs of such concentrations to interfere the apoptosis of Schwann cell (Ligustrazine hydrochloride and Astragalus polysaccharide are the main active ingredients of Tangtongfang). The experiment groups included the control group, high glucose group, Tangtongfang group, Ligustrazine group and Astragalus polysaccharide group. AV/PI double staining method was used to observe the apoptosis rate in each group. TUNEL method was used to observe the morphology of apoptotic cells. Western blot and RT-PCR were used to observe the expression changes of the apoptosis related mRNAs and proteins (bcl-2、bax、 Caspase-3、Caspase-9、Cytochrome C、Fas、FasL). The effect of Tangtongfang and its main active ingredients in interfering Schwann cell apoptosis induced by high glucose was observed and compared in many aspects.Results:1. In this study, we successfully incubated the primary Schwann cells with the purity degree reaching85%by combining the methods of tissue block culture, differential adhesion and digestion with low-concentration trypsin. It can be concluded from practice that the primary Schwann cells incubated in this method show such advantages as high purity degree, superior activity and good reproducibility which laid the foundation for the following study.2. In this study, the apoptosis condition of Schwann cells was determined by AnnexinV/PI double staining and Flow cytometry with different high glucose concentrations, different interfering time and different cell culturing densities. After many repeated experiments. It was finally confirmed that the most appropriate apoptosis model of Schwann cell was established during48-72h after high glucose interference.3. It was confirmed through CCK-8method that the drug containing serum can significantly reverse the proliferation inhibition of Schwann cells caused by high glucose. The main active ingredients of Tangtongfang:Obvious proliferation enhancement of Schwann cells was observed with Astragalus polysaccharide from160mg/L to3200mg/L and Ligustrazine hydrochloride from4mg/L to200mg/L, and the proliferation effect was promoted along with the extension of time. Then, for each ingredient, three interfering concentrations of high, medium and low were selected within the respective effective concentration to act on the Schwann cells incubated with high glucose. The results further confirmed that Astragalus polysaccharide and Ligustrazine hydrochloride can promote the proliferation of Schwann cells and improve the inhibition caused by high glucose.4. AnnexinV/PI double staining method and TUNE1immunofluorescence staining method were used to determine the effects of Tangtongfang and its main active ingredients on the apoptosis of Schwann cells induced by high glucose. The results showed that Schwan cells suffered significant apoptosis after being induced by high glucose for72h. Compared with the controlled group, the high glucose group and Ligustrazine group showed the most significant apoptosis (P<0.05). Astragalus polysaccharide group and Tangtongfang group showed higher apoptosis rates than the controlled group, but they were significantly lower than that of the high glucose group (P<0.05). No significant difference was noted between the Tangtongfang group and the Astragalus polysaccharide group, but the difference between Tangtongfang group and Ligustrazine group was significant(P<0.05).5. Western blot test showed that the bil-2/bax protein ratio of all interference groups was significantly lower than that of the controlled group (P<0.01). This indicated that anti-apoptotic protein was dominant in cells of the controlled group and the cells were protected. However, pro-apoptotic protein was overexpressed in other interference groups induced by high glucose, leading to apoptosis. The interference groups induced by high glucose showed increased expression of such proteins as Fas, FasL, Cytochrome C, Caspase-9, Caspase-3and ICAM-1with the most significant increase found in the high glucose group. The Tangtongfang group showed the minimum increase of such proteins compared with the controlled group (P<0.01or P<0.05), and the increases in the Ligustrazine group and Astragalus polysaccharide group were larger than the controlled group but smaller than the Tangtongfang group.6. RT-PCR experiment showed similar mRNA expression of apoptosis related genes and factors in both Tangtongfang group and the controlled group. Compared with the controlled group, the high glucose group showed significant decreased expression of bcl-2mRNA (P<0.01) and significant increased expression of bax, Cytochrome C, Caspase-9, Caspase-3, ICAM-1mRNA(P<0.01). The mRNA expression amount of the apoptosis related genes and factors in the Ligustrazine group and Astragalus polysaccharide group were between the Tangtongfang group and the high glucose group.Conclusion:With the major effects of supplementing qi and nourishing yin, promoting blood circulation and removing blood stasis, Tangtongfang can inhibit the influence of high glucose condition on the apoptosis of Schwann cells through regulating the activation and expression of the apoptosis related genes, factors and ICAM-1. In this way, peripheral nerve will be protected and diabetic peripheral neuropathy can be prevented and treated. Ligustrazine hydrochloride and Astragalus polysaccharide also show some effects of inhibiting the apoptosis of Schwann cells induced by high glucose to some degree independently. This indicates that the qi supplementing drug and blood circulation promoting drug in prescription serve a significant part in treatment, but Chinese herbal compound demonstrates superior effect than that of single herb prescription.