Pharmaceutical composition and application thereof

Abstract

The invention provides a pharmaceutical composition, which is prepared from rutin, hyperoside, isoquercitrin, gossypoin-8-O-beta-D-glucuronide, myricetin, quercetin-3 '-O-glucoside and quercetin according to the mass ratio of (0.0 to 0.3): (0.2 to 3.5): (0.2 to 3.5): (0.0 to 3.5): (0.0 to 1.8): (0.0 to 3.0): (0.0 to 0.9). The pharmaceutical composition has the effects of protecting podocytoskeleton and inhibiting inflammation, and has a good treatment effect on diabetic nephropathy.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition and also relates to the use of the pharmaceutical composition. Background Art

[0002] The structural formulas of quercetin-8-O-β-D-glucuronide, hyperoside, isoquercetin, myricetin, quercetin, quercetin-3′-O-glucoside, and rutin are as follows:

[0003]

[0004] The above compounds belong to flavonoid compounds, and some of them have been reported to have the effect of treating diabetes. However, it is still unknown what combination ratio of them can achieve the best effect, and it is necessary to develop a flavonoid combination with better efficacy. Summary of the invention

[0005] The invention provides a pharmaceutical composition and application thereof, which are found to have better therapeutic effect on diabetic nephropathy and can inhibit inflammatory response.

[0006] An object of the present invention is to provide a pharmaceutical composition comprising the following components in parts by weight:

[0007]

[0008] The pharmaceutical composition further contains the following components in parts by weight:

[0009] .

[0010] The pharmaceutical composition further contains the following components in parts by weight:

[0011] .

[0012] The pharmaceutical composition further contains the following components in parts by weight:

[0013]

[0014] or,

[0015]

[0016] or,

[0017]

[0018] or,

[0019]

[0020]

[0021] or,

[0022]

[0023] or,

[0024]

[0025] Another aspect of the present invention provides the use of the pharmaceutical composition in the preparation of a drug for treating inflammation and protecting podocytes, or the use of the pharmaceutical composition in the preparation of a drug for treating and inhibiting inflammatory damage to podocytes.

[0026] In yet another aspect of the present invention, there is provided use of the pharmaceutical composition in preparing a medicament for treating diabetic nephropathy.

[0027] The pharmaceutical composition provided by the present invention can upregulate synaptopodin and nephrin proteins to varying degrees, wherein S300 has the best upregulation level of synaptopodin and nephrin in podocytes. When the ratio of rutin: hyperoside: isoquercetin: gossypol-8-O-β-D-glucuronide: myricetin: quercetin-3′-O-glucoside: quercetin is 0:1.2:0.88:0.88:0.48:2.08:0.4, the protective effect on podocyte skeleton markers is the best.

[0028] The pharmaceutical compositions provided by the present invention can all reduce the mRNA expression level of TNF-α. It is speculated that when the ratio of rutin: hyperoside: isoquercetin: gossypol-8-O-β-D-glucuronide: myricetin, quercetin-3′-O-glucoside, and quercetin is 0.08: 1.56: 2.61: 0.98: 0.33, 1.28, and 0.14, the anti-inflammatory effect on podocytes is optimal. DETAILED DESCRIPTION

[0029] Example 1 Preparation of pharmaceutical composition

[0030] Take rutin, hyperoside, isoquercetin, hibifolin, myricetin, quercetin-3'-o-glucoside, and quercetin raw materials, weigh each active ingredient according to the weight ratio in the table below, mix, and prepare pharmaceutical compositions S1 to S21.

[0031]

[0032] Example 2 Preparation of pharmaceutical composition

[0033] Take rutin, hyperoside, isoquercetin, hibifolin, myricetin, quercetin-3′-o-glucoside, and quercetin raw materials, weigh each active ingredient according to the weight ratio in the table below, mix them, and prepare pharmaceutical compositions S100, S200, and S300.

[0034]

[0035] Example 3 Preparation of pharmaceutical composition

[0036] Take rutin, hyperoside, isoquercetin, hibifolin, myricetin, quercetin-3′-o-glucoside, and quercetin raw materials, weigh each active ingredient according to the weight ratio in the table below, mix, and prepare pharmaceutical compositions N1, N2, and N3.

[0037]

[0038] Example 4 Preparation of pharmaceutical composition

[0039] Take the raw materials of hyperoside, isoquercetin, hibifolin and quercetin-3'-o-glucoside, weigh each active ingredient according to the weight ratio in the following table, mix them, and prepare the pharmaceutical composition HIHQ.

[0040] Element Hyperoside Isoquercetin hibifolin Quercetin-3'-O-glucoside HIHQ 1.2 0.88 0.88 2.08

[0041] Example 5 Study on the effect of drug composition on podocyte skeleton markers and inflammation

[0042] 5.1 Experimental cells

[0043] Mouse podocytes (MPC5) were purchased from Beijing Beina Chuanglian Biotechnology Research Institute.

[0044] 5.2 Reagents, test drugs and instruments

[0045] Table 5-1 Reagents and test drugs

[0046]

[0047] Table 5-2 Instruments and Equipment

[0048]

[0049]

[0050] 5.3 Experimental Grouping:

[0051] Normal group (NG): 5.5 mM glucose

[0052] Hypertonic group (MA): 5.5 mM mannitol + 24.5 mM glucose

[0053] Model group (HG): 30 mM glucose

[0054] Positive drug (TFA): 30mM glucose + TFA

[0055] Negative control group (S0): 30 mM glucose + substrate

[0056] Each pharmaceutical composition group of Example 1: 30 mM glucose + S1 to S21

[0057] 5.4 Solution preparation

[0058] 5.4.1 PBS preparation

[0059] Weigh 8 g NaCl, 0.2 g KCl, 0.2 g KH2PO4, and 2.08 g Na2HPO4·12H2O, add 800 mL ultrapure water to dissolve, mix well, adjust the pH to 7.4, make up to 1 L, sterilize, add sterile water to 1 L, and store at 4°C for use.

[0060] 5.4.2 Preparation of 0.1% TritonX-100

[0061] Take an appropriate amount of TritonX-100 and dissolve it in PBS solution, dilute it 1000 times, mix it well, and use it immediately.

[0062] 5.4.3 Preparation of RPMI-1640 complete medium with 10% FBS

[0063] Take 44.5 mL of RPMI-1640 incomplete medium, add 5 mL of serum and 500 μL of HEPES, mix well, filter, and store at 4°C for later use.

[0064] 5.4.4 Preparation of RPMI-1640 starvation medium with 2% FBS

[0065] Take 24 mL of RPMI-1640 incomplete medium and PMI-1640 sugar-free medium, add 1 mL of serum and 500 μL of HEPES, mix well, filter, and store at 4°C for later use.

[0066] 5.4.5 Preparation of 30 mM high glucose medium

[0067] Add 24.5 mM glucose to RPMI-1640 starvation medium containing 2% FBS, fully dissolve, mix, filter, and store at 4°C for later use.

[0068] 5.4.6 Preparation of 30 mM mannitol solution

[0069] Add 24.5 mM mannitol to RPMI-1640 starvation medium containing 2% FBS, fully dissolve, mix, filter, and store at 4°C for later use.

[0070] 5.4.7 Preparation of drug stock solution

[0071] Weigh about 160 mg of each drug group, add 1 mL of DMSO, and vortex to dissolve to obtain a total flavonoid stock solution with a concentration of 160 mg / mL, then dilute it with DMSO to solutions with concentrations of 5, 10, 20, 40, and 80 mg / mL, and dilute it to the corresponding concentration using RPMI-1640 starvation medium containing 2% FBS.

[0072] 5.4.8 Preparation of 4% paraformaldehyde solution

[0073] Weigh 4 g of paraformaldehyde and dissolve it in 100 mL of PBS solution. Prepare it in a glass bottle, wrap it with tin foil and place it in a constant temperature incubator at 60°C overnight until the powder is completely dissolved. Refrigerate it at 4°C or -20°C and use it up within one week if it is freshly prepared.

[0074] 5.4.9 Preparation of 5% BSA solution

[0075] Weigh 5 g of BSA and dissolve it in 100 mL of PBS. Prepare it for immediate use.

[0076] 5.4.10 Preparation of MTT solution

[0077] Weigh 5 mg of MTT powder and prepare a storage solution with a concentration of 5 mg / mL in PBS. After sterilization by filtration through a 0.22 μm filter membrane, store at 4°C in the dark.

[0078] 5.5 Experimental modeling: MPC5 cells were stimulated with 30 mM glucose and the cell survival rate was detected. If the cell survival rate was (63.05±3.908)%, the model was successful and was used for subsequent experiments.

[0079] 6 Methods

[0080] 6.1 MPC5 cell recovery

[0081] Preheat the water bath to 37°C, take out the mouse podocytes (MPC5) frozen in the liquid nitrogen tank, and place them in a water bath to thaw quickly. Under sterile conditions, transfer the cells to a centrifuge tube, add RPIM-1640 complete medium containing serum, and centrifuge (800rpm, 4min). Discard the supernatant, resuspend the cells in a culture dish, add fresh culture medium, blow and evenly, place in a cell culture incubator (temperature 37°C, CO2 concentration 5%) for culture, and observe the cell growth after 12h.

[0082] 6.2 Subculture of MPC5 cells

[0083] When the cells grow to 80%-90% confluence, discard the original culture medium, wash with PBS solution 2-3 times, add 2mL trypsin digestion, and observe under a microscope. When the cells become round and the gaps become larger, add complete culture medium to stop digestion, gently blow to make a cell suspension, resuspend the cells in new culture medium, distribute at 1:3, move to cell culture dishes, add 3mL fresh culture medium to each dish, blow evenly, place in an incubator for culture, and observe cell growth after 12 hours.

[0084] 6.3 Cryopreservation of MPC5 cells

[0085] Take cells in the logarithmic growth phase, discard the original culture medium, wash 2-3 times with PBS, and add 2mL trypsin digestion (same as the previous step of subculturing). After centrifugation, resuspend the cells in the pre-prepared freezing solution (90% serum + 10% DMSO), 1-1.5mL per tube, and mark the cell name, growth generation, freezing date and other information, place in a program freezing box, freeze at -80℃ overnight, and transfer to a liquid nitrogen tank for long-term storage.

[0086] 6.4 Cell viability evaluation

[0087] MTT method was used for detection. MPC5 cells in the logarithmic growth phase were taken and inoculated into 96-well plates at 5000 cells / well. After being cultured overnight in a 37°C, 5% CO2 incubator, the supernatant was discarded. The cells were divided into a blank control group and a drug-treated group, with 6 replicates in each group. RPIM-1640 culture medium was added to the blank control group, and drug solutions of different concentrations (mass concentrations were 5, 10, 20, 40, 80, and 160 μg / mL, respectively) were added to the drug-treated group, 150 μL per well. After 48 hours, 0.5 mg / mL MTT solution was added at 100 μL / well. After 2-3 hours, the supernatant was discarded, 100 μL of DMSO was added to each well, and the absorbance (A) value of each well was detected at a wavelength of 490 nm using an enzyme reader, and the cell survival rate (%) was calculated according to the following formula: Cell survival rate (%) = (A drug-treated group / A blank control group) × 100%.

[0088] 6.6 Immunofluorescence assay to determine the effects of drugs on podocyte cytoskeleton markers

[0089] Take MPC5 cells grown to 80%-90%, adjust the cell density to 5000 and evenly inoculate them in 96-well plates. After incubation at 37°C and 5% CO2 overnight, remove the supernatant and group them according to the experiment. Add 150 μL of 1640 medium containing 5.5 mM glucose to each well of the control group (NG), add 150 μL of 1640 medium containing 30 mM glucose to the high glucose group (HG), and add 150 μL of a mixed solution of 5 μg / mL TFA and 30 mM glucose to the positive control group. Add 150 μL of a mixed solution of drugs with a final concentration of 5-10 μg / mL and 30 mM glucose to the S1-S21 drug administration groups. After treatment of each group of cells, place them in a 37°C, 5% CO2 incubator for 48 hours, remove the medium, add 4% paraformaldehyde, 60 μl / well, and fix at room temperature for 20 minutes. Then add 0.1% Tritonx-100 to permeabilize for 10-15 minutes, use 5% BSA to block for 1 hour, and after blocking, add 50μL / well (96 wells) of primary antibody solution to the cell solution and incubate overnight at 4°C. After the primary antibody incubation is completed, aspirate the primary antibody solution, add 50μL / well of AlexaFluor 488 conjugate anti-rabbit fluorescent secondary antibody solution to the cells, and incubate at room temperature in the dark for 1 hour. Add Hoechst 33342 to stain the nucleus at room temperature in the dark for 5-10 minutes, and observe the fluorescence under a fluorescence microscope.

[0090] 6.7 ELISA method to determine the effect of flavonoid monomers and combinations on podocyte inflammatory injury

[0091] Take MPC5 cells grown to 80% to 90%, adjust the cell density to 5000 and evenly inoculate them in 96-well plates. After culturing overnight in a 37°C, 5% CO2 incubator, remove the supernatant, and group them according to the experiment. The control group (NG) added 150 μL of 1640 medium containing 5.5 Mm glucose to each well, the high glucose group (HG) added 150 μL of 1640 medium containing 30 mM glucose, the positive control group (S22) added 150 μL of a mixed solution of total flavonoids with a concentration of 5 μg / mL and 30 mM glucose, and the negative control group (S23) added 150 μL of a mixed solution of a negative sample with a concentration of 1.16 μg / mL and 30 mM glucose. The S1-S21 drug administration groups added 150 μL of a mixed solution of drugs with a final concentration of 5-10 μg / mL and 30 mM glucose. After treatment, the cells in each group were placed in a 37°C, 5% CO2 incubator for 48 h. Synaptopodin and nephrin were determined by immunofluorescence, and TNF-α and IL-1β were determined according to the instructions of the ELISA kit.

[0092] 6.8 Data Processing

[0093] All data are expressed as mean ± standard deviation Statistical analysis was performed using GraphPad Prism 8.0.2, and one-way analysis of variance was used. P < 0.05 was considered statistically significant.

[0094] 7 Results

[0095] The results of immunofluorescence assay for synaptopodin and nephrin (fluorescence microscope NIB600, Nanjing Jiangnan Yongxin Optics Co., Ltd.) are shown in Table 1 below:

[0096]

[0097]

[0098] Note: *Compared with NG group, P<0.05; #Compared with model group, P<0.05; ##Compared with model group, P<0.01; ###Compared with model group, P<0.001; nsCompared with model group, there was no statistical significance.

[0099] Immunofluorescence was used to detect the effects of different drug groups on high glucose-induced podocyte cytoskeleton markers. The results showed that compared with the NG group (5.5 mM glucose), the HG group (30 mM glucose) downregulated synaptopodin and nephrin proteins, and other drug groups could also upregulate synaptopodin and nephrin proteins to varying degrees.

[0100] ELISA kits were used to detect the effects of different drug groups on high-glucose-induced podocyte inflammatory factors. The results showed that compared with the NG group (5.5mM glucose), the levels of TNF-α and IL-1β in the HG group (30mM glucose) increased; compared with the HG group, the drugs could reduce TNF-α and IL-1β to varying degrees. Among them, S11, S12, and S13 have the effect of significantly downregulating TNF-α and IL-1β to normal levels at the same time. Among them, the S1, S2, S3, S8, S11, and S13 drug-administered groups have the effect of significantly downregulating TNF-α and IL-1β to normal levels at the same time, and S1, S2, and S13 have the effect of significantly adjusting the four factors synaptopodin, nephrin, TNF-α, and IL-1β to normal levels at the same time.

[0101] Example 6 Further study of podocyte skeleton markers

[0102] Cell model: MPC5 cells

[0103] Experimental membrane preparation and method: same as above.

[0104] Experimental groups: Normal group (NG): 5.5 mM glucose

[0105] Hypertonic group (MA): 5.5 mM mannitol + 24.5 mM glucose

[0106] Model group (HG): 30 mM glucose

[0107] Positive drug group (TFA): 30 mM glucose + TFA

[0108] Example 2 Pharmaceutical composition: 30 mM glucose + S100-S00

[0109] After treatment, the cells in each group were placed in a 37°C, 5% CO2 incubator for 48 hours, and synaptopodin and nephrin were determined by immunofluorescence (fluorescence microscope Leica, Leica, Germany). The test results are as follows

[0110] As shown in Table 1:

[0111] Group Synaptopodin nephrin NG 0.0468±0.0021 0.0482±0.0031 MA <![CDATA[0.0388±0.0050 ns ]]> <![CDATA[0.0410±0.0049 ns ]]> HG <![CDATA[0.0323±0.0038 *** ]]> <![CDATA[0.0348±0.0033 *** ]]> TFA <![CDATA[0.0440±0.0052 # ]]> <![CDATA[0.0493±0.0076 # ]]> S100 <![CDATA[0.0430±0.0062 ns ]]> <![CDATA[0.0490±0.0066 # ]]> S200 <![CDATA[0.0472±0.0064 # ]]> <![CDATA[0.0507±0.0052 ## ]]> S300 <![CDATA[0.0428±0.0030 ## ]]> <![CDATA[0.0417±0.0068 ns ]]>

[0112] *Compared with NG group, P<0.05; #Compared with model group, P<0.05; ##Compared with model group, P<0.01; nsCompared with model group, no statistical significance.

[0113] The optimized flavonoid monomers were used for experiments to detect their effects on podocyte skeleton markers synaptopodin and nephrin. The results showed that there was no significant difference in the MA group (5.5mM glucose + 24.5mM mannitol) compared with the NG group (5.5mM glucose); the synaptopodin and nephrin proteins in the HG group (30mM glucose) were downregulated, and the total flavonoid group and each drug group could upregulate synaptopodin and nephrin proteins to varying degrees, among which S300 achieved the best upregulation level of synaptopodin and nephrin in podocytes. It is speculated that the protective effect on podocyte cytoskeleton markers is optimal when the ratio of rutin: hyperoside: isoquercetin: gossypol-8-O-β-D-glucuronide: myricetin: quercetin-3′-O-glucoside: quercetin is 0:1.2:0.88:0.88:0.48:2.08:0.4.

[0114] Example 7 Further study of podocyte inflammatory factors

[0115] Cell model: MPC5 cells

[0116] Experimental modeling: same as above

[0117] Experimental groups:

[0118] Normal group (NG): 5.5 mM glucose

[0119] Hypertonic group (MA): 5.5 mM mannitol + 24.5 mM glucose

[0120] Model group (HG): 30 mM glucose

[0121] Positive drug group (TFA): 30 mM glucose + TFA

[0122] Example 3 Pharmaceutical composition: 30 mM glucose + N1 to N3

[0123] After treatment, the cells in each group were placed in a 37°C, 5% CO2 incubator for 48 hours, and TNE-α was determined by RT-qPCR. The test results are shown in Table 1 below (fluorescence microscope Leica, Leica, Germany):

[0124] Group TNF-α NG 1.0150±0.2034 MA <![CDATA[0.9678±0.3077 ns ]]> HG <![CDATA[2.143±0.1868 ** ]]> TFA <![CDATA[0.5192±0.1381 ### ]]> N1 <![CDATA[1.552±0.1405 # ]]> N2 <![CDATA[0.7418±0.2619 ## ]]> N3 <![CDATA[1.4730±0.9681 # ]]>

[0125] Note: *Compared with NG group, P<0.05; #Compared with model group, P<0.05; ##Compared with model group, P<0.01; ###Compared with model group, P<0.001; nsCompared with model group, there was no statistical significance.

[0126] RT-qPCR was used to detect the effects of various drugs on high glucose-induced podocyte inflammatory factors. The results showed that compared with the NG group (5.5 mM glucose), there was no significant difference in the MA group (5.5 mM glucose + 24.5 mM mannitol); the mRNA expression of TNF-α in the HG group (30 mM glucose) increased; compared with the HG group, each drug group could reduce the mRNA expression level of TNF-α. It is speculated that when the ratio of rutin: hyperoside: isoquercetin: gossypol-8-O-β-D-glucuronide: myricetin, quercetin-3'-O-glucoside, quercetin is 0.08:1.56:2.61:0.98:0.33, 1.28, 0.14, the anti-inflammatory effect on podocytes is the best.

[0127] Example 6 Study on the mechanism of action of drug composition and TRPC6

[0128] Cell model: MPC5 cells

[0129] Experimental modeling: same as above

[0130] Experimental groups:

[0131] Normal group (NG): 5.5 mM glucose

[0132] Hypertonic group (MA): 5.5 mM mannitol + 24.5 mM glucose

[0133] Model group (HG): 30 mM glucose

[0134] Positive drug group (TFA): 30 mM glucose + TFA

[0135] Example 4 Pharmaceutical composition: 30 mM glucose + HIHQ

[0136] After treatment, cells in each group were placed in a 37°C, 5% CO2 incubator for 48 h, and TRPC6, RhoA, and ROCK1 were measured by RT-qPCR, and TRPC6 and synaptopodin were measured by immunofluorescence. The test results are as follows

[0137] As shown in Table 1:

[0138] Group TRPC6 RhoA ROCK1 NG 1.0310±0.3725 1.0030±0.0886 0.6600±0.1676 MA <![CDATA[0.7198±0.4746 ns ]]> <![CDATA[1.0530±0.2217 ns ]]> <![CDATA[0.7160±0.1805 ns ]]> HG <![CDATA[3.2090±0.5122 ** ]]> <![CDATA[2.0080±0.1187 *** ]]> <![CDATA[2.9480±0.7306 * ]]> TFA <![CDATA[0.9584±0.2299 ## ]]> 1.7970±0.2824 <![CDATA[0.8960±0.2797 # ]]> HIHQ <![CDATA[0.5864±0.4438 ## ]]> 1.8650±0.2394 <![CDATA[0.7006±0.2945 # ]]>

[0139] Note: *Compared with NG group, P<0.05; **Compared with NG group, P<0.01; ***Compared with NG group, P<0.001; #Compared with model group, P<0.05; ##Compared with model group, P<0.01; nsCompared with model group, no statistical significance.

[0140] The effects of TFA and HIHQ on the expression of TRPC6, RhoA, and ROCK1 in podocytes induced by high glucose were detected by real-time quantitative PCR. The results showed that compared with the NG group, the MA group had no effect on the mRNA expression levels of TRPC6, RhoA, and ROCK1 in podocytes, and the expression levels of TRPC6, RhoA, and ROCK1 in podocytes in the HG group were significantly increased; after TFA and HIHQ intervention in podocytes, the expression of TRPC6 and ROCK1 was significantly reduced, and the expression level of RhoA did not change significantly.

[0141] Example 7 Effects of TRPC6 inhibitors on podocyte cytoskeleton markers

[0142] Cell model: MPC5 cells

[0143] Experimental modeling: same as above

[0144] Experimental groups: Normal group (NG): 5.5 mM glucose

[0145] Hypertonic group (MA): 5.5 mM mannitol + 24.5 mM glucose

[0146] Model group (HG): 30 mM glucose

[0147] Positive drug group (TFA): 30 mM glucose + TFA

[0148] SAR group: 30 mM glucose + SAR

[0149] TFA+SAR: 30mM glucose+TFA+SAR

[0150] HIHQ+SAR: 30mM glucose+HIHQ+SAR

[0151] Example 4 Pharmaceutical composition (HIHQ): 30 mM glucose + HIHQ

[0152] After treatment, the cells in each group were placed in a 37°C, 5% CO2 incubator for 48 hours, and TRPC6 and synaptopodin were determined by immunofluorescence. The test results are shown in Table 1 below:

[0153] Group TRPC6 Synaptopodin NG 0.1293±0.0390 0.1597±0.0253 MA <![CDATA[0.1618±0.0155 ns ]]> <![CDATA[0.1442±0.0175 ns ]]> HG <![CDATA[0.2345±0.0257 *** ]]> <![CDATA[0.0860±0.0075 ** ]]> SAR <![CDATA[0.1015±0.0210 ### ]]> <![CDATA[0.1503±0.0210 ## ]]> TFA <![CDATA[0.1577±0.0312 # ]]> <![CDATA[0.1115±0.0061 ## ]]> HIHQ <![CDATA[0.1473±0.0335 ## ]]> <![CDATA[0.1197±0.0063 ### ]]> TFA+SAR <![CDATA[0.1230±0.0171 ### ]]> <![CDATA[0.1308±0.0093 ### ]]> HIHQ+SAR <![CDATA[0.1202±0.0227 ### ]]> <![CDATA[0.1318±0.0068 ### ]]>

[0154] Note: **P<0.01 compared with NG group; ***P<0.001 compared with NG group; #P<0.05 compared with model group; ##P<0.01 compared with model group; ###P<0.001 compared with model group; ns, no statistical significance compared with model group. Immunofluorescence method was used to determine the effects of TRPC6 inhibitor on podocyte TRPC6 and synaptopodin. Compared with NG group, there was no significant change in TRPC6 and synaptopodin in MA group, while the expression level of TRPC6 in HG group increased significantly, and the expression level of synaptopodin decreased significantly. After 48h of drug intervention, compared with HG group, the expression levels of TRPC6 in SAR group, TFA group, HIHQ group, TFA+SAR group, and HIHQ+SAR group decreased to varying degrees; the expression level of synaptopodin increased. Compared with the SAR group, the TRPC6 in the TFA+SAR group and the HIHQ+SAR group increased slightly, and the synaptopodin decreased slightly, but the difference was not statistically significant (P>0.05). Compared with the TFA group and the HIHQ group, the TRPC6 in the TFA+SAR group and the HIHQ+SAR group decreased slightly, and the synaptopodin increased slightly, but the difference was not statistically significant. The results showed that both TFA and HIHQ could reduce the expression level of TRPC6 in podocytes induced by high glucose and increase the expression of synaptopodin, but the inhibitory effect of SAR7334 on TRPC6 and the protective effect on synaptopodin could be affected by TFA and HIHQ, which may be related to the pathway of TFA action, and its specific action process needs further study.

[0155] Conclusion: The combinations of rutin, hyperoside, isoquercetin, quercetin-3′-O-glucoside and quercetin with quercetin as the mother core, and the combinations of hyperoside, isoquercetin, quercetin-3′-O-glucoside and hibifolin based on the content of ingredients have a strong correlation with synaptopodin and nephrin skeleton proteins, and can be used as alternative drugs for the pharmacological substances with protective effects on the podocyte skeleton. The combination of hyperoside, isoquercetin and hibifolin has a strong correlation with the inhibition rate of TNF-α and IL-1β, and can be used as an alternative drug for the pharmacological substances that inhibit inflammatory damage of podocytes.

Claims

1. A pharmaceutical composition comprising the following components in parts by weight:

2. The pharmaceutical composition according to claim 1, comprising the following components in parts by weight:

3. The pharmaceutical composition according to claim 1, comprising the following components in parts by weight:

4. The pharmaceutical composition according to claim 1, comprising the following components in parts by weight: or, or, or, or, or, Alternatively, the weight ratio of rutin: hyperoside: isoquercetin: gossypol-8-0-β-D-glucuronide: myricetin: quercetin-3′-O-glucoside: quercetin is 0:1.2:0.88:0.88:0.48:2.08:0.4, Alternatively, the weight ratio of rutin: hyperoside: isoquercetin: gossypol-8-0-β-D-glucuronide: myricetin, quercetin-3′-O-glucoside, quercetin is 0.08:1.56:2.61:0.98:0.33, 1.28, 0.

14.

5. Use of the pharmaceutical composition according to claim 1 in the preparation of a drug for treating and inhibiting inflammatory damage of podocytes.

6. Use of the pharmaceutical composition according to claim 1 in the preparation of drugs for treating inflammation and protecting podocytes.

7. Use of the pharmaceutical composition according to claim 1 in the preparation of a medicament for treating diabetic nephropathy.

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