| Background and ObjectiveChronic Kidney Disease (CKD) is a clinical common, frequently-occurring and difficult disease. Currently the incidence of CKD among global adult is as high as 8%-16%, while the awareness is lower than 10%, and 1%-3% of these CKD patients will be converted to end stage renal disease (ERSD). As many CKD patients pay insufficient attention for early prevention and the risk factors control, along with the accelerating of the aging population, the increasing of chronic infectious diseases, the increasing prevalence of hypertension and diabetes, the morbidity of CKD and its induced ERSD are increasing every year, which brings a heavy financial burden to society and family because of its poor prognosis and high cost. CKD has become a worldwide problem threatening human health.At present, the main clinical treatment strategies include:control blood pressure, control blood glucose for diabetes patients, reduce proteinuria, application of antioxidants, control blood lipid, control hyperhomocysteinemia, improve anemia, prevent infection, prevent hypokalemia and high hyperphosphatemia, correct water and electrolyte and acid-base balance disorders, control diet and so on. The main mechanisms of these treatments are controlling inflammatory response, reducing the level of oxidative stress, inhibiting the expression of fibrosis-related factors, thereby delaying the progress of CKD.However, the existing treatments are aiming at controlling the risk factors for CKD, protecting kidney function and reducing complications. Although the basis of the lesion has been static, kidney function declines continuously, and a large number of CKD patients cannot afford the expensive long-term medical treatment cost. So, searching for an effective and cheap conservative therapy to improve the symptoms of CKD, stable residual renal function, postpone their entry into "renal replacement therapy", and improve the life quality of patients, are major issue to be solved for clinical medical workers. Integrative medicine has unique advantages and potentials in this regard. Uremic clearance granules (UCG) developed by our hospital just came out under such background, which being the first Chinese herbal medicine on prevention of CKD progress in this field in China. Because of its better efficacy and relatively lower price, it has climbed to the top sales in China nephropathy oral medicine market for many years. Based on problems and deficiencies existing in the production and promotion of UCG, our research group performed the secondary development of UCG on the basis of UCG, and eventually obtained Huang Gan Formula (HGF) under downsizing and reorganizing of the original prescription, which aimed to further improve the product quality, enhance the efficacy and clarify the mechanism.Our previous study showed that:HGF could significantly reduce the level of serum creatinine (Scr), blood urea nitrogen (BUN), inhibit oxidative stress, and delay the progress of renal fibrosis in the chronic renal failure rat models prepared by adenine. This study is aimed to establish the HPLC fingerprint and to analyze the complex chemical composition of HGF on qualitative and quantitative by using HPLC-QTOF/MS technique, hopefully to clarify the pharmacodynamic substance basis of HGF. And then construct the CKD rat models by 5/6 nephrectomy, further verifying the efficacy of HGF, and to study the impact of HGF on Wnt/β-catenin signaling pathway associated proteins in vivo and in vitro experiments, to investigate the possible mechanisms of anti-renal fibrosis, and to provide new ideas and reliable experimental basis for the clinical treatment of CKD.Methods(1) Using ethanol extraction and water extraction by alcohol sedimentation combinational techniques, to prepare the HGF extract. Ten batches of HGF extract were used to establish the fingerprint by HPLC. Similarity indices of the HPLC chromatograms of ten batches of extracts were evaluated by the professional software, Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version 2004A), which was recommended by Chinese Pharmacopoeia Commission and State Food and Drug Administration (SFDA). Then to qualitative analyze the main components of HGF by HPLC-QTOF/MS method, and to verify the qualitative results by reference standards, and finally to measure the percentage of the main components by establishing a HPLC standard curve.(2) Using 5/6 nephrectomy to construct CKD rat model, the levels of Scr and BUN in the 5/6 nephrectomy model group and the sham group were determined before treatment (2 weeks after operation) to test whether the model was successful. After the models succeed, we divided the experimental rats into 8 groups according to the weight stratified random method,11 to 12 rats each group. Define them as follows:the model group (Model), Uremic clearance granules group (UCG), losartan group (Losartan), HGF low-dose group (HGF-L), HGF middle dose group (HGF-M), HGF high-dose group (HGF-H), sham-operated group (Sham) and background control group (BC). Drug delivery methods:UCG was given a gavage of 3.6g/kg/d premixing solution, Losartan was given a gavage of 30 mg/kg/day, The HGF-L, HGF-M and HGF-H groups were given a gavage of 3.6,7.2 and 14.4g/kg/d separately. HGF-L was clinical equivalent dose group. Gave medicine twice a day. Sham and Model groups were given an equal volume of distilled water. BC group was given a middle dose of HGF suspension. All groups continuously administrated for 12 weeks. Collected the 24h urine after the last administration, and collected the serum through the abdominal aorta under anesthesia, and determined the levels of Scr, BUN,24h urinary protein (UPr), urine creatinine (Ucr), total cholesterol (CHOL), low-density lipoprotein (LDL) and other biochemical markers using automatic biochemical analyzer. Collected remnant kidney in each experimental group, and placed one half in 4% paraformaldehyde fixing solution after longitudinally section of kidney tissues used for pathological and immunohistochemical detection. The other half of the cortex was divided into three portions. One part for the preparation of tissue homogenate and measurement of the malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-PX) content in renal tissue. The other two parts frozen at-80℃ for RT-PCR, western blot and other experiments.(3) Using rat renal tissues to extract RNA and total protein. Detected the mRNA expression levels of Wntl, β-catenin, Tcf4, Gsk-3β, Dkkl and Fnl by RT-PCR, and detected the renal tissue protein lever of each experimental group by western blot. Paraffin-embedded tissue prepared for the kidney section, to analyze the protein expression levers of Wntl, β-catenin, Tcf4, Gsk-3β, Dkkl and Fnl in renal tissue parts by immunohistochemistry and semi-quantitative their expressions.(4) Establishing the epithelial-mesenchymal transition (EMT) model in HK-2 cells:HK-2 cells were induced by high glucose medium (D-glucose 30mmol/L) for 12h,24h and 48h separately, and used ordinary medium and mannitol isotonic medium as controls, and then detected the expression of Alpha smooth muscle actin (a-SMA) and β-catenin proteins by western blot. HGF with different concentrations was added to the HK-2 cells for 48h to detect the proportion of apoptotic cells and dead cells by flow cytometry, which aimed to screen the maximum safe concentration of HGF. Wnt signal pathway associated proteins detection:the experimental groups include ①normal control group (NC),5.5mmol/L glucose concentration in 2% FBS culture medium,②high glucose model group (HG), 30mmol/L glucose concentration in 2% FBS culture medium, ③HGF intervention group prepared with high glucose, HGF100 (100μg/ml, the low-dose group) and HGF200 (200μg/ml, the high-dose group), ④CG-001 positive control group (10μM). The cells in each group were collected after culture 12h,24h and 48h, RT-PCR and western blot were performed to detect the expression levers of Wntl, P-catenin, Tcf4, a-SMA and Fnl. Prepared the cell climbing slides for the same treatment above, and then to observe the expression level of β-catenin and a-SMA under confocal microscope by immunofluorescence.Results1. Substance basis of HGF(1)1g dried HGF extract was equivalent to 4.6g original herbal medicine. HGF appeared maximum absorption peak at 254nm under PDA detector full wavelength scanning from 200nm to 400nm, which showed an obvious characteristic peaks and good peak separation. According to Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version 2004A), the control fingerprint generated from 10 batches of HPLC chromatogram, where a total of 21 common peaks were marked. The similarities of each batch fingerprint were 0.929,0.987,0.982,0.965,0.993,0.974,0.994,0.997,0.942 and 0.981. All the similarities were above 0.92, which indicated that the 10 batches of HGF had a good consistency and a stable extraction method. Using UV HPLC fingerprint could control the overall quality of HGF well.(2) Using HPLC-QTOF/MS, we obtained 38 compounds in response from MS total ion current by automatically searching and manual matching methods, of which 11 chemical compositions in positive ion modes,27 chemical ingredients in negative ion modes. By the comparative analysis of chromato graphic retention times with standards and the comparative analysis of MS molecular ion peak and fragment peak, we identified 8 major compounds, which were liquiritin, tectoridin, luteolin, aloe-emodin, rhein, emodin, chrysophanol and physcion.(3) Using HPLC standard curve, we measured 8 main components of HGF. They and their contents were:liquiritin (0.167%), tectoridin (1.98%), luteolin (1.03%), aloe-emodin (3.78%), rhein (3.13%), emodin (1.54%), chrysophanol (2.72%) and physcion (1.55%).2. The pharmacodynamics research of HGF(1) Two weeks after modeling, the Scr and BUN levels in nephrectomy rats were significantly higher than the sham group (.P<0.001), which indicated the 5/6 Nx models were successful. During administration, the rat weight of each experimental group had no significant differences, and showed a gradual upward trend.(2) The biochemical indicators in serum:the experimental data presented normal distribution in each group, so used one-way ANOVA for multiple comparisons between groups. Statistical results showed that:12 weeks after administration, the rat kidney index (RI), Scr, BUN and UPr in model group were significantly higher than the sham group and the BC group (P<0.01), while creatinine clearance (Ccr) was significantly lower than the sham group and the BC group (P<0.05). There were no significantly differences in each biochemical indicator between the sham group and the BC group (P>0.05). The RI, Scr, BUN, Ccr, UPr levels among experimental groups were significantly different (F=4.044, P=0.001; F=58.951, P<0.001; F=114.147, P<0.001; F=4.113, P-0.001; F=6.108, P<0.001):the RI levels in each treatment group was significantly lower than the model group (P<0.001), the BUN levels in HGF low and high dose groups were significantly lower than the model group (P<0.05), and the HGF three groups were also lower than losartan group (P<.01). The Scr levels in the middle and high-dose groups were significantly lower than the model group (P<0.05), whereas the Ccr levers in HGF low and high dose groups were significantly higher than the model group and the losartan group (P<0.05). The UPr levels in HGF three groups were significantly lower than model group (P<0.05), but the low and high dose groups were significantly higher than losartan group (P<0.01). There had no significant difference of the RI, Scr, BUN, Ccr, UPr levels between HGF three dose groups and UCG group (P>0.05).(3) The CHOL and LDL levels in each treatment group were significantly lower than the model group (P<0.05), except for the CHOL level in losartan group (P= 0.971). The HGF three dose groups significantly reduced level of MDA in renal tissue, and increased the level of SOD, T-AOC, CAT and GSH-PX, which had statistically significance with the model group (P<0.05). The Serum AOPP levels of HGF low, medium and high dose groups were significantly lower than the model group (P<0.001), and in the middle and high dose groups were significantly lower than the UCG group (P<0.001) and the losartan group (P< 0.01).(4) HE and Masson staining showed that:15 weeks after operation, the sham and BC groups had no pathological changes in renal tissue. There had significantly different degrees of ease in inflammatory cell infiltration, tubular atrophy, glomerular sclerosis, interstitial fibrosis and other symptoms in HGF groups.3. The effect of HGF on Wnt/β-catenin signaling pathway in CKD rat renal tissue(1) RT-PCR results showed that:there had statistical differences of Wntl, β-catenin, Tcf4, Gsk-3β, Dkk1, and Fnl mRNA expression levels among each experimental group of renal tissue in rats (F=3.784, P<0.01; F=12.942, P<0.001; F=9.781, P<0.001; F=7.004, P<0.001; F=8.059, P<0.001; F=2.858, P=0.021). HGF low, medium and high dose groups significantly inhibited the mRNA expressions of Wntl, β-catenin, Tcf4 and Fnl, and the differences had statistically significant (P<0.05). Meanwhile, the three doses of HGF groups could raise the mRNA levels of Gsk-3β and Dkkl, compared with the sham group and the background control group (P<0.01,P<0.05)).(2) Western blot results coincided with the RT-PCR results:Compared with the model group, HGF low, medium and high dose groups significantly inhibited the expression of Wntl (P<0.001), β-catenin (P<0.05), Tcf4 (P<0.05) and Fnl (P<0.001), while there were no significantly difference of the expression of Wntl, P-catenin, Tcf4, Dkkl, Fnl and the phosphorylate levels of Gsk-3β protein between the sham group and the background control group (P> 0.05).(3) Immunohistochemistry results showed that:there all had the protein expressions of Wntl,β-catenin, Tcf4, Gsk-3β, Dkkland Fnl in the sham group and the model group, but with a small range, light color, relatively weak expression in the sham group. The proteins expression sites of Wntl, Dkkl and Tcf4 were similar, mainly in the renal tubular epithelial cells and the renal glomerular mesangial cells, with a highly expression in the glomeruli. The expression sites of β-catenin and Gsk-3β were mainly in the renal glomerular mesangial cells and the distal convoluted tubule epithelial cells. While Fnl primarily expressed in the renal glomerular mesangial cells, the basement membrane and the renal tubular, which was the main reason for the tube interstitial fibrosis. The semi-quantitative results broadly consistented with the RT-PCR and the western blot results.4. The interventional effect of HGF on EMT in HK-2 cells through Wnt pathway(1) The maximum safe concentration to HK-2 cell for 48h treatment of HGF was 200ug/ml. After high glucose induced HK-2 cells for 12h,24h and 48h, the expression of a-SMA, a EMT mark protein, and the expression of β-catenin, a key protein of Wnt pathway, gradually increased, but there was no obvious expression for the HM group and the normal control group (NC) within the treatment time. All that illustrated that high glucose could induce the occurrence of EMT in HK-2 cells, and activate the expression of Wnt pathway associated proteins, while the high osmotic pressure of high glucose had no effect on the expression of the target proteins.(2) RT-PCR results showed that:high glucose induced HK-2 cells for 12h,24h and 48h, and given drug treatment at the same time, the mRNA expressions of Wntl, β-catenin, Tcf4, a-SMA and Fnl were weak before 12h, and the inhibition of the drug was not obvious. During 24h-48h, the activation of Wnt pathway got to the top. Statistical results showed that:high glucose induced HK-2 cells for 48h, the mRNA expression level of Wntl in high glucose model group (HG) was significantly higher than in the normal group (P<0.001), ICG-001 positive control group could significantly inhibited its expression (vs. HG P<0.001), HGF high and low dose groups also had the inhibitory effect (P=0.01, P=0.02), and had no significant difference with the ICG-001 group (P=0.276, P=0.122). However, there was no significant difference between the HGF low-dose group and the HGF high-dose group (P=0.601). And the mRNA expressions of β-catenin, Tcf4, a-SMA and Fnl in each dose group exhibited the same inhibitory trend with Wntl, and the difference was statistically significant (P<0.05).(3) Western blot results showed that:induced HK-2 cells for 48h with high glucose, Wnt pathway associated proteins significantly up-regulated, the secretions of a-SMA and Fnl increased. The ICG-001 positive control and HGF high and low dose groups could inhibit the expression of proteins related to this pathway, reduce the secretion of a-SMA and Fnl in EMT process in HK-2 cells, the differences were statistically significant. There had the same phenomena in immunofluorescence experiments, which the HGF high and low dose groups could reduce the expression of a-SMA and p-catenin.Conclusions(1) The preparation of HGF extract is stable and reliable, and different batches of HGF have good consistency. The overall quality of HGF can be well controlled through the establishment of HPLC fingerprint.(2) Substance basis of HGF are clarified by quantitative and qualitative analysis of major components using Q-TOF/MS and HPLC methods, which improves the quality control and standard level of HGF.(3) HGF can effectively protect the 5/6 nephrectomy rats remnant kidney function, significantly reduce the serum level of Scr, BUN and urine protein in CKD model rats, and improve the filtration function and anti-oxidative stress capacity in the remnant kidney, then reduce the level of renal fibrosis and effectively delay the progression of CKD.(4) HGF inhibits Wnt/β-catenin signaling pathway in 5/6 nephrectomy rats remnant kidney, and may reduce renal fibrosis by this.(5) HGF can inhibit the activation of Wnt/β-catenin pathway in HK-2 cells induced by the high glucose. The interruption of EMT process by HGF may associate with inhibition of this pathway. |
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