| Glomerular sclerosis is the main pathological basis of chronic renal failureand the final outcome of various glomerular diseases, with chronic renal failureas the clinical performance. And the main pathological features include theproliferation of mesangial cell (MC) and excessive accumulation of extracellularmatrix (ECM), which can lead to the formation of glomerular sclerosis. Anincreasing number of basic and clinical researches regarding traditional Chinesemedicine and western medicine treatments for glomerular sclerosis have obtainedgreat achievements, mainly focusing on the glomerular sclerosis mechanisms.There is little evidence about the treatment. Many scholars are trying to explorevarious measures for preventing glomerular sclerosis, delaying diseasedevelopment, and retarding renal failure. Single Chinese herbs, single herbalextracts and Chinese herbal compound prescription can inhibit MC proliferation,reduce ECM accumulation, antagonize adverse effects caused by cytokines, preventand treat glomerular sclerosis.Previous studies of our research group have investigated the change of MCsin rats after treated with separated prescriptions of Yushen granules, includingone for benefiting qi, removing stasis and relieving toxicity, one for benefitingqi and removing stasis, and one for benefiting qi and relieving toxicity.Preliminary results showed that, compound No.1that was consisted of Astragalus,Danshen Root and Oldenlandia could significantly inhibit the MC proliferation,decrease the ECM synthesis, and prevent glomerular sclerosis. According to ahypothesis that “stagnant toxin blocks kidney channel" raised by ProfessorZhang Jun, the pathogenesis of various preliminary and secondary glomerulardiseases attributes to the deficiency-and toxin-caused blood stasis. This studyaims to observe the efficacy and mechanisms underlying small compoundprescriptions (Astragalus, Danshen Root and Oldenlandia) for benefiting qi,detoxification and removing stasis on the glomerular sclerosis, in a broader attempt to provide theoretical and practical significance for the preventionand treatment of glomerular sclerosis.Experiment1: Influence of compound prescription and itssingle herbs on MC proliferation and apoptosis in ratsPurpose:The aim of this study was to discuss the effect of compound prescriptionand its single herbs on the angiotensin II (Ang II)-induced MC proliferationand apoptosis in rats.Material and method:1. Main materials: Sixty Wistar rats, weighing200±20g； rat MCs； Chineseherbal medicine compound consisting of Astragalus, Danshen Root and Oldenlandia；three single herb groups: Astragalus, Danshen Root and Oldenlandia,respectively；western medicine control group: telmisartan；Dulbecco’s modifiedEagle’s medium (DMEM) powder, fetal bovine serum, Ang II, MTT, AnnexinV-FITC,trypan blue.2. Main instruments: Superclean bench； CO2thermostatic incubator； invertedfluorescence microscope； Benehmark microplate reader； electronic precisebalance； ultrasonic cell grinder； flow cytometry； protein nucleic acidspectrophotometer； electrophoresis system； EPS-300(Tanon, Shanghai, China)；gelgraph analyzing system； high-speed freezing centrifuge； DHG series heatingand drying oven； glass Dounce homogenizer； cell culture flask； cell cultureplate (96-well,12-well)； centrifuge tube, frozen pipe, microporous filter.3. Serum pharmacological method: Traditional Chinese medicine preparations:decoction pieces of Chinese herbs were immersed in cold water for30minutesand boiled in strong fire for30minutes, then the herbal liquid was filteredand the herbs were boiling with water for additional20minutes. High-doseprescriptions can be obtained after three times of decoctions. Astragalusdecoction was condensed to0.7g/mL, Danshen Root decoction was condensed to 0.25g/mL, Oldenlandia decoction was condensed to0.3g/mL, compound decoctionwas condensed1.25g/mL, and telmisartan concentration was adjusted into0.6mg/mL.Drug serum preparation: Sixty Wistar rats were randomly divided into six groups,with ten rats in each group, namely normal group, compound group, Astragalusgroup, Danshen Root group, Oldenlandia group, and western medicine control group(telmisartan). Rats of the normal group, Chinese herb groups, and telmisartangroup were respectively administrated with4mL of distilled water andcorresponding drug liquids, twice per day, for3consecutive days. At1hourafter the last administration, the abdominal aorta blood was sampled andcentrifuged at1500r/min for5minutes, to obtain blood serum. The complementwas deactivated in56℃water bath for30minutes, filtered with0.22-μm sterilefilter, stored at-20℃for further use.4. MC culture: The rat MC morphology was observed under inverted microscope.Results showed that, MCs were well growing and spindle-shaped, with a clear ovalnucleus in the center, cells adhered to the wall of culture flask and the majorityof cells were fused.1/2-2/3of the culture medium was replenished with DMEMcontaining10%fetal bovine serum, and the cells were incubated at37℃,5%CO2humidity. The culture medium was changed every2-3days.5. Random grouping:Ang II group: Culture medium was added with10-6mol/L Ang II and DMEM containing10%fetal bovine serum；Normal serum group: Culture medium was added with10-6mol/L Ang II and10%normalrat serum with no Chinese herb.Compound prescription group: Culture medium was added with10-6mol/L Ang IIand10%serum of rats treated with compound prescription.Astragalus group: Culture medium was added with10-6mol/L Ang II and10%serumof rats treated with Astragalus.Danshen Root group: Culture medium was added with10-6mol/L Ang II and10%serumof rats treated with Danshen Root. Oldenlandia group: Culture medium was added with10-6mol/L Ang II and10%serumof rats treated with Oldenlandia.Western medicine control group: Culture medium was added with10-6mol/L AngII and10%serum of rats treated with telmisartan.6. Detection of MC proliferation and apoptosis: The absorbance value at570nmwas determined with ELISA colorimetric assay, using MTT method. The rat MCs at1×104/mL were seeded onto96-well culture plate, with200μL in each well. Therewere six parallel wells in each group. The adherent cells were cultured withserum-free DMEM for additional24hours, and the majority of cells weresynchronized in G0/G1phase and cultured with10%normal rat serum and10%serumcontaining drugs,200μL in each well, for48hours. Then culture medium wasremoved and each well was supplemented with20μL MTT6hours prior to thecompletion of culture. After the cultivation was completed, the MTT solutionwas removed, and each well was added with150μL dimethyl sulfoxide, shakenat37oC microporous oscillator for10minutes. After all crystallizations werecompletely dissolved, the absorbance value was read with ELISA assay at570nm.Flow cytometry was used to determine the effect of drug-contained serum on theMC proliferation and apoptosis.Results:The proliferation and apoptosis of rat MCs showed extremely significantdifferences in Ang II group comparison with normal control group (P<0.01),suggesting that Ang II can promote abnormal proliferation of rat MCs, and MCproliferation model is successfully established. There were significantdifferences between Ang II group and various drug groups (P<0.01), whichindicated that all the drug interventions can inhibit the Ang II-induced MCproliferation. The compound group also showed significant differences incomparison to other drug groups (P<0.01or P<0.05). This is evidence that thecompound prescription has the strong inhibition effect on MC proliferation. Nosignificant difference was found between any of other drug groups. The efficacy of drug-contained serum was similar at24hours and48hours, which clearlydemonstrated that the inhibition effect on the MC proliferation was not dependenton the time. Flow cytometry results showed that, the apoptosis rate was increasedsignificantly compared with normal control group and Ang II group (P<0.05), withno significant difference between drug groups (P>0.05).Conclusion:The compound prescription of benefiting qi, detoxification and removing stasisis superior to single Chinese herbs on inhibiting the MC proliferation andpromoting the MC apoptosis, thus can prevent and treat glomerular sclerosis,as well as protect the kidney.Experiment2: Influence of compound prescription and itssingle herbs on fibronectin and collagen type IV proteinlevels in rat MCsPurpose:The aim of this study was to observe the effect of compound prescription andits single herbs on fibronectin and collagen type IV protein levels inextracellular matrix of rat MCs.Material and method:1. Main materials: Serum collagen type IV ELISA kit (Beijing Bangding TaikeBiotechnology Co., Ltd., Beijing, China), serum fibronectin ELISA kit (BeijingBangding Taike Biotechnology Co., Ltd., Beijing, China), collagenase type IV (Sigma,USA).2. Main methods: The MC supernatant was determined using double antibody ELISAsandwich assay.100μL culture medium was taken out and the absorbance valuewas detected with a microplate reader according to the instructions of ELISAkit for serum fibronectin and collagen type IV. The absorbance value=(absorbance value of tested well–absorbance value of normal controlled well). Results:The serum fibronectin and collagen type IV protein levels had extremelysignificant differences between Ang II group and normal control group (P <0.01),indicating Ang II can lead to a significantly increased level of fibronectinand collagen type IV proteins, promote ECM synthesis, and accelerate glomerularsclerosis formation. There were also significant differences between Ang IIgroup and drug groups (P <0.05), which suggested that Chinese herbal medicineprescriptions can significantly decrease level of fibronectin and collagen typeIV proteins, inhibit the ECM synthesis, and prevent glomerular sclerosis. Thechanges in the compound prescription group were significantly different withthose in telmisartan group and other single herbal groups (P <0.05). The compoundprescription has the strongest effect to down-regulate the serum levels offibronectin and collagen type IV protein, and delay glomerular sclerosis.Conclusion:The compound prescription of benefiting qi, detoxification and removing stasiscan effectively reduce levels of fibronectin and collagen type IV protein anddecrease ECM synthesis in MCs, and the compound is superior to single herbs onthe prevention of glomerular sclerosis.Experiment3: Influence of compound prescription and itssingle herbs on matrix metalloproteinases (MMPs) and tissueinhibitor of metalloproteinases (TIMPs) in rat MCsPurpose:The aim of this study was to investigate the effect of compound prescriptionand its single herbs on MMP3and TIMP1genetic expression in rat MCs, and topreliminarily reveal the underlying mechanism.Material and method:1. Main materials and equipments: Trizol, isopropyl alcohol, chloroform, DNA-MARKER, RT-PCR kit, MMP-3, TIMP-1. Ultra-cold freezer； ultra-pure water；electric glass homogenate machine； ultrasonic cell grinder； protein nucleicacid spectrophotometer； deep-freeze refrigerator； superclean bench； swiftmixing； PCR instrument； electrophoresis system； electrophoresis tank；microwave； solid-liquid phase hybridization instrument.2. Methods: RT-PCR assay was used for the detection of each drug-contained serumon the MMP3and TIMP1mRNA expression in rats.Results:The MMP-3and TIMP1mRNA expression greatly varied in different groups. All drugtreatments could significantly promote the MMP3mRNA expression and decreasethe TIMP1mRNA expression, especially the compound prescription group. This isevidence that the compound prescription can reduce ECM synthesis and preventglomerular sclerosis, and the effect is associated with the increased MMP3mRNAexpression and the decreased TIMP1mRNA expression.Conclusion:The compound prescription of benefiting qi, detoxification and removing stasisinhibits the MC proliferation and reduces the ECM synthesis, which contributesto the decrease of MMP3mRNA expression and the increase of TIMP1mRNA expression.Experiment4: Influence of compound prescription and itssingle herbs on rat MCsPurpose:The aim of this study was to investigate the effect of compound prescriptionand its single herbs on MC signaling pathway Smad3/Smad7mRNA genetic expressionin rats, and preliminarily reveal the underlying mechanism.Material and method:1. Main materials: Smad3and Smad7reagent.2Main methods: The signal transmission of Smad3/7in MCs was detected. TheSmad3/7mRNA expression was also determined. Results:The effect of each drug treatment on the intercellular signaling pathwaySmad3/Smad7mRNA expression in rats was investigated. Results showed that, thecompound prescription and its single herbs can significantly inhibit Smad3expression and promote Smad7expression, and the compound was superior to singleherbs. So compound prescription can suppress the TGF-beta/Smad signaling pathwayin the MCs, thus delay glomerular sclerosis formation. This is the geneticexplanation for the mechanism underlying compound prescription inhibits MCproliferation and ECM accumulation, as well as protects the kidney.Conclusion:The compound prescription of benefiting qi, detoxification and removing stasissuppresses the TGF-beta/Smad signaling pathway in the MCs, thereby delaying theformation of glomerular sclerosis. We have revealed the underlying mechanismsassociated with compound prescription inhibiting MC proliferation and ECMaccumulation, as well as protecting the kidney in the gene and protein levels. |
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