Prevention And Treatment Of Immunosuppressive Injury In Diabetic Nephropathy With Tangshenqing No.2 And Its Clinical Curative Effect Analysis

Objective:The experimental research part is aimed to explore the molecular mechanism of TSQ No.2 through regulating AMPK signaling pathway in inhibiting DN inflammatory injury by culturing cells and establishing animal model methods.The clinical research part is aimed to assess clinical efficacy and safety of TSQ No.2 for DN Ⅲ、Ⅳ patients with deficiency of kidney-Yin, phlegm-blood stasis, heat and toxin syndrome pattern.Methods:Vivo Experimental Research:SPF KK-Ay mice were used to induce DN models.The mice were feed with high fat diet for 3 weeks.Then to detect blood glucose, and 24h urine protein to evaluate if the models were successful. Mice were be regarded as the successful models if its blood glucose was more than or equal to 13.9 mmol/L, and 24h urinary protein higher than normal group.30 successful DN models were divided into model group randomly, positive drug group, high dose of Chinese medicine group, Middle dose of Chinese medicine group and low dose of Chinese medicine group, each group with 6 mice,with 6 male C57BL/6J mice were designed as normal control group.Mice in Chinese medicine groups were treated with TSQ No.2 in 40.66g·kg-1·d,20.33 g·kg-1·d,10.17g·kg-1·d dosage by gavage,mice in positive drug group received valsartan tablets 13.33 g·kg-1·d,mice in control group and model group were gavaged witn deionized water. Mice in each group were treated by lavage once daily,12-week period continuous. After 12 weeks,the specimens of serum and renal tissue were reserved,which were used to detect factors as follows:RT-PCR analysis method was used to detect transcription level of AMPKa1,ADPN,NF-KB p65,iNOS,MCP-1,TNF-α,IL-6 mRNA in renal tissue;Western blot analysis method were used to detect protein expression levels of AMPKal,ADPN,NF-κB p65,iNOS; ELISA analysis method was used to detect the protein expression levels of IL-6,MCP-1,TNF-α; Immunohistochemistry analysis method were used to detect the protein expression levels of AMPKα1,ADPN,NF-κB p65 and iNOS. Vitro Experimental Research:To culture the mouse glomerular mesangial cell line SV40 MES 13 in 5%CO2,37℃ constant temperature incubator. Experimental groups:control group(D-Glucose 5.6mmol/L),high glucose group(D-Glucose30mmol/L),high glucose+TSQ 5μmol/L group,high glucose+TSQ10μmol/L group,high glucose+TSQ 20μmol/L group.Cells in all groups were cocultured 12h,24h,36h.Then Inflammation-Related Cytokines were measured as follows:RT-PCR analysis method was used to detect the transcription level of AMPKα1、IκBa and TLR4 mRNA; Western Blot analysis method was used to detect the protein expression levels of AMPKα1,lκBα and TLR4.Clinical Research:To collect 80 cases of DN Ⅲ、Ⅳ patients with deficiency of kidney-Yin, phlegm-blood stasis, heat and toxin syndrome pattern. All of the cases were in accordance with the inclusion criteria,exclusion criteria and rejection criteria.All cases were randomly divided into treatment group or control group.The patients in treatment group were taking TSQ No.2 decotion,one dose each day.While the patients in control group were taking valsartan in dose of 80mg each day based on their basis treatments. Treatment time was 3 months.To record the patient’s name, gender, age, height, weight, course of disease, using drugs,complications and other information before treatment in detail. To record changes of clinical examination indexes,clinical symptoms, tongue and pulse before and after treatment.To analysis and discuss on clinical curative effect of TSQ No.2 according to curative effect evaluation standard,to evaluate the changes of clinical curative effects and safety indexes,TCM syndrome integral changes between two groups after the end of treatment.Results:Vivo Experimental Research:RT-PCR results:Compared with normal group,the transcriptional levels of anti-inflammatory cytokines AMPKal mRNA and ADPN mRNA were significantly decreased in model group(P<0.01).Compared with model group,transcriptional levels of AMPKal mRNA increased significantly in positive medicine group,high-dose and middle-dose Chinese medicine groups(P<0.01);the transcriptional levels of ADPN mRNA had significant difference in positive drug group and middle-dose Chinese medicine groups (P< 0.01, P<0.05).Compared with the normal group,the transcriptional level of proinflammatory cytokine NF-κB p65,IL-6,TNF-a,iNOS,MCP-1mRNA were significantly increased in model group(P<0.01).Compared with the model group,the transcriptional level of NF-κB p65,IL-6,TNF-a,MCP-1mRNA were down-regulated statistically in positive medicine group and Chinese medicine groups(P<0.01);the transcriptional level of iNOS mRNA was significantly down-regulated in positive drug group,high-dose and middle-dose Chinese medicine groups(P < 0.01,P< 0.05). Western Blot and ELISA test results:compared with the normal group,the protein expression levels of anti-inflammatory cytokines AMPKal, ADPN had significant statistical difference in model group(P<0.01).Compared with the model group,the protein expression level of AMPKal was significantly increased in positive medicine group and Chinese medicine groups(P<0.01,P<0.05);the protein expression levels of ADPN was significantly increased in positive medicine group(P< 0.01).Compared with the normal group,the protein expression levels of proinflammatory factors NF-κBP65,IL-6,TNF-α,iNOS,MCP-1 had significant statistical difference in model group(P<0.01). Compared with the model group, the protein expression levels of NF-κB p65,IL-6,TNF-α,MCP-1 were down-regulated in positive medicine group and traditional Chinese medicine groups (P<0.01);the protein expression levels of iNOS were down-regulated in positive drug and high-dose Chinese medicine groups(P<0.01,P<0.05). Immunohistochemical results:compared with the normal group, the protein expression level of AMPKal and ADPN were down-regulated in model group (P< 0.01), the protein expression level of NF-κB p65 and iNOS was significantly increased in model group (P< 0.01, P< 0.05). compared with the model group, the protein expression level of AMPKal was significantly increased in all drug groups (P< 0.01); the protein expression level of ADPN was increased in positive drug group,high-dose and low-dose Chinese medicine groups (P< 0.01, P< 0.05); the protein expression level of NF-κB p65 was decreased in positive drug group and high-dose Chinese medicine groups (P< 0.01); the protein expression level of iNOS was down-regulated in positive drug group,high-dose and low-dose Chinese medicine groups (P< 0.05). Vitro Experimental Research:RT-PCR results:compared with normal control group,the transcription level of AMPKal mRNA was down-regulated at 12h and 36h in high glucose group(P< 0.05);the transcription levels of IκBα mRNA had statistical difference at 12h and 24h in high glucose group(P<0.01,P<0.05);the transcription levels of TLR4 mRNA at 36h was significantly increased in high glucose group(P< 0.01).Compared with high glucose group,the transcription level of AMPKal mRNA was up-regulated at 12h and 36h in TSQ 5μmol/L group(P<0.01,P<0.05),the transcription level of IKBa mRNA increased significantly at 24h(P<0.05);the transcription level of AMPKα1 mRNA at 12h significantly increased in TSQ 20μmol/L group(P<0.01),the transcription level of IKBa mRNA was up-regulated at 12h in TSQ 20umol/L group(P<0.05);The transcription level of TLR4 mRNA in each drug group were significantly down-regulated in 36h(P<0.01).Western blot results:compared with normal control group,the protein expression level of AMPKal and IκBα were down-regulated in high glucose group between 12h and 36h(P<0.01,P<0.05);the protein expression level of TLR4 increased significantly at 12h and 36h in high glucose group (P< 0.01).Compared with the high glucose group,the protein expression level of AMPKα1 and IκBα were up-regulated between 12h and 36h in TSQ5μmol/L group(P<0.01,P<0.05);the protein expression level of AMPKal was up-regulated at 12h and 36h in TSQ 10μmol/L group(P<0.01,P<0.05),the protein expression level of IKBa was upregulated between 12h and 36h (P<0.01),the protein expression level of TLR4 decreased at 12h in TSQ 1 0μmol/L group(P <0.01);the protein expression level of AMPKal significantly increased between 12h and 36h in TSQ 20μmol/L group(P<0.05),the protein expression level of IKBa upregulated at 24h(P< 0.05),the protein expression level of TLR4 had significant difference at 12h and 36h in TSQ 20μmol/L group(P<0.01).Clinical Research:we expected to collect 80 DN Ⅲ, Ⅳ patients with deficiency of kidney-Yin, phlegm-blood stasis, heat and toxin syndrome pattern. In fact,67 cases completed this clinical study. The patients’general information as follows: ① There were 34 cases in the TSQ No.2 treatment group,in which 17 cases of male,17 cases of female,with average age 55.88±7.61 years old.There were 33 cases in the Valsartan control group,in which 18 cases of male and 15 cases of female,with average age 56.08±6.54 years old.There were 31 cases in accordance with the diagnosis standard of DN Ⅲ,and 36 cases were in accordance with the DN Ⅳ diagnosis standard.The proportion of female patients in DN stage Ⅲ was higher than that of male,while the proportion of male in DN stage Ⅳ was higher than that of female.②the distribution of the tongue body in DN Ⅲ and Ⅳ stage:the dark red tongue was in highest frequency,followed by red or reddish tongue,purplish tongue. Some patients presented plump tongue, scanty fluid. Above informations suggested that the dark red tongue marking phlegm and blood stasis were common syndromes in DN Ⅲ-Ⅳ, red or pale red tongue was marking deficiency of Qi and blood,and less fluid performed heat and blood stasis.③the distribution of the coated tongue:the thin and yellowish fur was in the highest frequency in DN Ⅲ-Ⅳ tongue,followed by yellowish greasy tongue,white and greasy fur, thin and whitish fur, less fur and incomplete loss of the coating of the tongue marking deficiency of yin and body fluid.④ the distribution of pulse conditions:the taut and rolling pulse condition was in the highest frequency in DN Ⅲ-Ⅳ stage,followed by heavy and slippery pulse, thready pulse on behalf of yin deficiency syndrome.slippery pulse is more prominent.We can speculate the etiology and pathogenesis of DN is the deficiency Yin,with phlegm,dampness,heat and other symptoms performance. ⑤ the clinical evaluation of TSQ No.2:compared with situations before treatment,the levels of Scr,24h urinary protein and urinary albumin were decreased inTSQ No.2 group (P<0.05),the levels of TC,LDL decreased significantly,and had statistically significant difference (P<0.01, P<0.05).Compared with the control group,the levels of Scr,TC,TG,24h urinary protein,urinary albumin were significantly down-regulated in treatment group after the treatment(P<0.05), the total clinical effective rate was 73.53%.⑥ the TCM syndrome curative effect evaluation of TSQ No.2:the TCM syndrome score was decreased significantly in TSQ No.2 group (P<0.01),1 cases were marked improvement,25 cases were effectively improvement. The total effective rate was 76.47%. Fasting blood glucose, postprandial blood glucose and glycosylated hemoglobin in the two groups were controlled at a reasonable level. The safety indicators, such as blood routine test, urine routine test, stool routine test and liver function test showed no significant changes. During the treatment, no adverse events occurred in the TSQ No.2 group.Conclusion:TSQ No.2 can down-regulate the expression level of proinflammatory cytokine, effectively alleviate the DN renal tissue inflammatory immune pathological injury by playing the role of AMPK activator; TSQ No.2 can obviously improve the clinical symptoms of patients with DN, lower proteinuria, and protect renal function.