Effects Of The “tonifying, Clearing, And Eliminating Kidney Collaterals” Method On The Proliferation Of Human Glomerular Mesangial Cells Via The JAK2/STAT1 Pathwa

Purpose:Based on the theory of"renal networkopathy"in the theory of network disease,this study used serum pharmacological methods to conduct in vitro experiments to explore the inhibitory effect of the medicated serum of The method of"tonic,clearing,eliminating the kidney"on the proliferation of human mesangial cells（HMC）induced by angiotensin II through JAK2/STAT1 signaling pathway through JAK2/STAT1 signaling pathway,and explored the target and mechanism of action of The method of"tonic,clearing,eliminating the kidney"in the prevention and treatment of Ig A nephropathy,which is"renal network disease"The in-depth study of theoretical treatment of Ig A nephropathy provides theoretical basis and experimental data support.Material and method:1.Preparation of medicinal serum:40 SD-grade male rats were randomly divided into 5 groups,namely high-dose group of traditional Chinese medicine （high-dose The method of"tonic,clearing,eliminating the kidney"）,medium-dose group of Chinese medicine（medium-dose The method of"tonic,clearing,eliminating the kidney"）,low-dose group of Chinese medicine（low-dose The method of"tonic,clearing,eliminating the kidney"）,normal group,Western medicine group,8 in each group.The rats were reared adaptively for3 days,the amount of crude drug and the amount of gavage were calculated,and the decoction of Chinese medicine was prepared by the decoction room of the Chinese Medicine Bureau of the Affiliated Hospital of Liaoning University of Chinese Medicine,and the gastric gavage was continuously carried out for 7 days,and the abdominal aorta of each group was collected after the last dose.The blood of each group was transferred to a centrifuge for centrifugation,the supernatant was taken after centrifugation,transferred to a 56°C water bath for 30min,filtered and sterilized,and stored in a-20°C refrigerator for later use.2.Human mesangial cell culture and grouping:human mesangial cells were removed from the liquid nitrogen tank,inoculated into the culture flask,RPMI-1640 culture medium（containing10%fetal bovine serum）was added to the vial,and the culture flask was placed in a 37°C,5%CO₂incubator for culture,and the liquid was changed regularly.Through the induction of angiotensin II.,the proliferation of human mesangial cells was promoted,and the cells were divided into high-dose groups of traditional Chinese medicine,medium-dose group of traditional Chinese medicine,low-dose group of traditional Chinese medicine,normal group,western medicine group,pharmacological serum intervention western medicine group and various traditional Chinese medicine groups.3.Detection method:MTT method to detect the growth of mesangial cells in each group for48h;ELISA detected angiotensin II.-induced contents of p JAK2 and STAT1 in mesangial cells.Real-time PCR detected the relative expression levels of STAT1mRNA,SOCS1mRNA and IL-1βmRNA induced by angiotensin II.in mesangial cells.Results:1.The results of MTT method showed that compared with the normal group,the mesangial cells of the model group proliferated significantly（p<0.01）,indicating that angiotensin II was used as a stimulator to successfully induce HMC proliferation.Compared with the model group,the number of mesangial cells in the medium and high dose groups of traditional Chinese medicine was reduced（p<0.05）.2.The results of real-time PCR showed that:（1）SOCS1mRNA expression:SOCS1mRNA expression in the model group was significantly lower than that in the normal group（p<0.01）;compared with the model group,all traditional Chinese medicine groups were elevated（p<0.05）.Compared with the Chinese medicine groups,there were significant differences between the low-and medium-dose groups and the high-dose group（p<0.01）,and there was no significant difference between the low-dose group and the medium-dose group（p>0.05）.（2）STAT1mRNA expression:the expression of STAT1mRNA in the model group was significantly higher than that in the normal group（p<0.01）,and compared with the model group,the western medicine group and the traditional Chinese medicine group decreased（p<0.05）.The differences between the dose groups of traditional Chinese medicines were between the low-dose group of traditional Chinese medicines,the medium-dose group and the high-dose group（p<0.01）.（3）IL-1βmRNA expression:IL-1βmRNA expression in the model group was significantly higher than that in the normal group（p<0.01）,and compared with the model group,the western medicine group and the traditional Chinese medicine group decreased（p<0.05）.Compared with the Chinese medicine group,the differences between the low-dose group of traditional Chinese medicine,the medium-dose group and the high-dose group were detailed（p<0.01）.3.ELISA results showed that:（1）STAT1 expression:STAT1 expression in the model group was significantly higher than that in the normal group（p<0.01）,and the comparison between the traditional Chinese medicine group was reduced（p<0.05）.The differences between the dose groups of traditional Chinese medicines were between the low-dose group of traditional Chinese medicines,the medium-dose group and the high-dose group（p<0.01）.（2）p JAK2 expression:the expression of p JAK2 in the model group was significantly higher than that in the normal group（p<0.01）,and the difference between the low-dose group of traditional Chinese medicine,the medium-dose group and the high-dose group（p<0.01）was compared between the dose group of traditional Chinese medicine.Conclusion:1.The method of"tonic,clearing,eliminating the kidney"can significantly inhibit the increase in the number of cells caused by Ang II.stimulation of target cells（human mesangial cells）,and the effective targets may be JAK2,STAT1,SOCS1,IL-1β.2.The mechanism of action of The method of"tonic,clearing,eliminating the kidney"may be to inhibit p-JAK2 activation,reduce STAT1 production,inhibit JAK2/STAT1 activation,promote SOCS1 production in HMC,reduce IL-1βcontent,accelerate apoptosis of abnormal proliferative cells,and reduce and repair the pathological damage caused by Ig AN,so as to achieve the effect of preventing and treating Ig AN.3.Clarify the mechanism of Ig AN of The method of"tonic,clearing,eliminating the kidney"acting on Ig AN based on the principle of"supplementation,cleansing and elimination of kidney network",and enrich the application of"supplement,clear and eliminate kidney network"method in the prevention and treatment of Ig AN.