Diabetic nephropathy (DN) is a microvascular complication induced by diabetes mellitus (DM). It is the leading cause of end-stage renal disease (ESRD) and death in patients with DM [
Angiotensin-converting-enzyme inhibitors (ACEI)/angiotensin receptor blockers (ARB) are the only drug classes recommended by ADA for clinical use to control DN urinary albumin excretion and disease progression [
Abundant evidence has confirmed that insulin resistance is associated with the development and progression of DN [
In traditional Chinese medicine (TCM), “yin” and “yang” represent two different types of functions. “Yin” stands for inhibition and inactivation, and “yang” stands for promotion and stimulation; “yin” and “yang” can also be described as negative and positive feedback or regulatory networks [
The db/db mouse is an animal model for spontaneous type 2 DM. These animals are obese, hyperinsulinemic, and hyperglycemic [
Twenty-four male db/db mice (8 weeks old) and eight C57BL/6 mice (8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (certificate number: SCXK [Beijing] 2012-0001). This study was carried out in the animal laboratory with barrier facilities (certificate number: SYXK [Beijing] 2015-0001) of Dongzhimen Hospital Affiliated to Beijing University of Chinese Medicine. All animals were handled according to the guidelines of the Beijing University of Chinese Medicine Animal Research Committee. The experimental protocol was approved by the Laboratory Animal Welfare and Ethics Committee of Beijing University of Chinese Medicine (certificate number: 16-14). QDTS granules are composed of Dihuang (
After 4 weeks of adaptive feeding, all mice were divided into four groups according to blood glucose, body weight, and 24-h urinary albumin excretion rate (UAE): C57BL/6 group (as the normal control), db/db group (as the DN model group), db/db + valsartan group and db/db + QDTS group, 8 mice in each group. The mice were housed in cages (temperature 22-24°C, humidity 35-45%, under a 12-h light/dark cycle) and given standard diet and water
Body weight and random blood glucose were measured every two weeks. Blood glucose was measured by OneTouch® Ultra® Glucometer (Johnson & Johnson, USA) using the blood collected by cutting the tip of the tail. Fasting blood glucose and fasting serum insulin were measured by glucose oxidase and radioimmunoassay (XH-6020 automatic radioimmunoassay counter, Xi'an Nuclear Instrument Factory, China) separately at the end of the experiment. Homeostasis model assessment-insulin resistance (HOMA-IR) was calculated according to the following formula: fasting plasma glucose (FPG, mmol/L) × fasting insulin (FINS, mIU/L)/22.5.
UAE was measured every four weeks from the starting of the experiment. All of the mice were transferred into the metabolic cage (no feed or water was provided) and 24-h urine was collected. The total amount of the urine was measured, and after centrifugation (3000 rpm, 10 min), the supernatant was stored at −80°C. Urinary albumin was detected by using mice albumin ELISA (enzyme-linked immunosorbent assay) Kit (R&D systems, Minnesota, USA). UAE was defined as urinary albuminuria (
Cr, BUN, and UA were measured to assess the renal function. At the end of the experiment, blood samples were obtained by enucleating the eyeballs. Serum was isolated by centrifugation (3800 rpm, 10 min). The serum Cr, BUN, and UA were analyzed by automatic biochemical analyzer (Olympus AU480, Japan).
At the end of the experiment, the left kidney was removed, fixed in 4% paraformaldehyde (PFA), and embedded in paraffin. Sections about 2-3
Calculation of the percentage of renal fibrosis was conducted as described here: in each section, 10 randomly selected fields were observed under 200x magnification. The ratio of the blue areas of the collagen fibers to the total area was determined as a percentage in each field by Image Pro Plus 6.0 and the mean was calculated [
Proteins extracted from the kidney tissue (stored in liquid nitrogen) were separated on 10% SDS-polyacrylamide electrophoresis gels and then transferred to polyvinylidene fluoride (PVDF) membranes. After being blocked with 5% nonfat milk in TBST for 2 h, the PVDF membrane was incubated with the following primary antibodies overnight: IRS-1, IRS-1 (Ser307), Akt, p-Akt, ERK, and p-ERK antibody (CST, Massachusetts, USA); IRS-1 (Tyr896), PI3K, p-PI3K, p38MAPK, p-p38MAPK, JNK, and p-JNK antibody (abcam, Cambridge, UK); IRS-1 (Ser302) antibody (SAB, Maryland, USA). Next the membranes were washed with 1x TBST for three times, each time for 8 min and then incubated with biotin-labeled goat anti-rabbit or goat anti-mouse IgG (1:5000) at room temperature for 1 h. 5% milk was only used for nonphosphorylated primary antibodies; 3% BSA was used for membrane blocking and for dilution of all phosphorylated antibodies. The ECL hypersensitive luminescent liquid A and B (equal volume) was mixed, and the membrane was incubated with the mixed liquid for 2 min in the dark. The membrane was then placed in a gel imager for signal detection. The image was analyzed with Image Pro Plus 6.0.
All data are presented as mean ± standard error (SEM). Data were analyzed by SPSS22.0 statistical software. One-way analysis of variance was performed to test the differences between the groups. For groups having equal variances, the LSD (least significant difference) method was used; for those having unequal variances, Dunnett’s T3 was used.
In the present study, the body weight and the random blood glucose were measured every two weeks. The plasma fasting insulin and fasting blood glucose were measured at the end of the twelfth week (i.e., the end of the treatment), and HOMA-IR was calculated. The results showed that compared with the C57BL/6 mice, the body weight and the level of blood glucose of the db/db mice increased, and the HOMA-IR elevated significantly (
Comparison of body weight, blood glucose, and insulin resistance index of mice in different groups. (a) Body weight. (b) Homeostasis model assessment-insulin resistance (HOMA-IR). (c) Level of blood glucose. HOMA-IR = fasting plasma glucose × fasting insulin/22.5. C57BL/6, the normal control group; db/db, the diabetic nephropathy (DN) model group; db/db + v and db/db + valsartan, the valsartan intervention group; db/db + Q and db/db + QDTS, the QDTS granules intervention group. n = 8 for each group.
Microalbuminuria is the earliest clinical manifestation of DN, which will continuously advance to severe proteinuria and renal failure if not controlled properly. What is more, albuminuria is also an independent risk factor for accelerating the progression of DN [
Comparison of 24-h urinary albumin excretion rate (UAE) and renal function of mice in different groups. (a) 24-h UAE. (b) Serum creatinine (Cr) level. (c) Urea nitrogen (BUN) level. (d) Uric acid (UA) level. 24-h UAE was measured every four weeks and Cr, BUN, and UA were measured at the end of treatment. C57BL/6, the normal control group; db/db, the diabetic nephropathy (DN) model group; db/db + v and db/db + valsartan, the valsartan intervention group; db/db + Q and db/db + QDTS, the QDTS granules intervention group. n = 8 for each group.
At the end of the experiment, we measured the serum Cr, BUN, and UA levels. The results showed that there was no significant difference in serum Cr and BUN between groups (Figures
According to the HE staining, the morphological structure of the glomeruli and the renal tubules in the renal cortex was normal for mice in the C57BL/6 group. Masson’s trichrome staining showed that the blue areas of the collagen fibers mainly located in the renal tubular basement membrane, the mesangial region, and around the peritubular capillaries (Figure
Morphological changes of the kidney of mice in different groups. (a) HE and Masson’s trichrome staining of renal sections. (b) Percentage of areas showing fibrosis. Sections stained with Masson’s trichrome staining were used to calculate the percentage of areas showing renal fibrosis. At 200x magnification, 10 nonoverlapping fields showing the renal interstitium were selected in each section, the ratio of the blue areas indicating the collagen fibers to the total area was calculated, and the mean value of each treatment group was compared. C57BL/6, the normal control group; db/db, the diabetic nephropathy (DN) model group; db/db + v and db/db + valsartan, the valsartan intervention group; db/db + Q and db/db + QDTS, the QDTS granules intervention group. n = 3 for each group.
The renal tissue in the db/db group showed glomerular capillary loop hypertrophy and increased mesangial matrix, decreased Bowman's capsule cavity, and vacuolar degeneration of the renal tubular epithelial cells (Figure
The expression and phosphorylation levels of PI3K and Akt proteins in renal tissues were detected by western blot. The results showed that the expressions of PI3K as well as the phosphorylated forms of PI3K and Akt in renal tissues of the db/db mice were significantly decreased compared with the C57BL/6 mice (Figures
QDTS granules promoted the activation of PI3K/Akt signaling pathway in the kidney of the db/db mice. (a) Expression of PI3K. (b) Expression of phosphorylated PI3K. (c) Expression of Akt. (d) Expression of phosphorylated Akt. (e) Western blot of the expression of PI3K, phosphorylated PI3K, Akt, and phosphorylated Akt. Lane 1, the C57BL/6 group; Lane 2, the db/db model group; Lane 3, the db/db + v group; Lane 4, the db/db + Q group. C57BL/6, the normal control group; db/db, the diabetic nephropathy (DN) model group; db/db + v, the valsartan intervention group; db/db + Q, the QDTS granules intervention group. n = 4 for each group.
Since the phosphorylation of IRS-1 is the key process in insulin signaling [
QDTS granules reduced phosphorylation of the serine residues of IRS-1 and restored the phosphorylation balance of tyrosine/serine residues of IRS-1 in kidney tissues of the db/db mice. (a) Expression of IRS-1. (b) Expression of phosphorylated Tyr896 of IRS-1. (c) Expression of phosphorylated Ser302 of IRS-1. (d) Ratio of phosphorylated Ser302/Tyr896 of IRS-1. (e) Expression of phosphorylated Ser307 of IRS-1. (f) Ratio of phosphorylated Ser307/Tyr896 of IRS-1. (g) Western blot of the expression of IRS-1 and the phosphorylated tyrosine and serine residues of IRS-1. Lane 1, the C57BL/6 group; Lane 2, the db/db model group; Lane 3, the db/db + v group; Lane 4, the db/db + Q group. C57BL/6, the normal control group; db/db, the diabetic nephropathy (DN) model group; db/db + v, the valsartan intervention group; db/db + Q, the QDTS granules intervention group. n = 4 for each group.
ERK, p38MAPK, and JNK are the major members of the mitogen-activated protein kinase (MAPK) family. It has been shown that MAPK family members can promote the phosphorylation of the serine residues of IRS-1 and as a result inhibit the phosphorylation of the tyrosine residues of IRS-1 [
QDTS granules attenuated the activation of the MAPK signaling pathway in the kidney of the db/db mice. (a) Expression of ERK. (b) Expression of phosphorylated ERK. (c) Expression of p38MAPK. (d) Expression of phosphorylated p38MAPK. (e) Expression of JNK. (f) Expression of phosphorylated JNK. (g) Western blot of the expression of EPK, phosphorylated ERK, p38MAPK, phosphorylated p38MAPK, JNK, and phosphorylated JNK. Lane 1, the C57BL/6 group; Lane 2, the db/db model group; Lane 3, the db/db + v group; Lane 4, the db/db + Q group. C57BL/6, the normal control group; db/db, the diabetic nephropathy (DN) model group; db/db + v, the valsartan intervention group; db/db + Q, the QDTS granules intervention group. n = 4 for each group.
The db/db mouse is an animal model for spontaneous type 2 DM derived from a single autosomal recessive mutation of the leptin receptor gene. Thus, the db/db mice show the characteristic obesity and enormous appetite; hyperinsulinemia, insulin resistance, and hyperglycemia occur at the age of 2-4 weeks; the secretory function of
There are three signaling pathways activated by leptin, including JAK/STAT (Janus kinase/signal transducer and activator of transcription), MAPK, and PI3K (phosphatidylinositol 3-kinase) pathways. As described before, PI3K is also one of the main downstream pathways of insulin signaling. Leptin resistance may cause an increase in insulin requirement and insulin resistance [
The MAPK signaling pathway mainly consists of the extracellular-signal regulated protein kinase (ERK), p38MAPK, and c-Jun amino-terminal kinase (JNK) pathways. It has been suggested that there may be some connections between the MAPK and the PI3K/Akt signaling pathways. Various studies indicated that the three MAPK signaling pathways could inhibit the transduction of insulin signaling by different mechanisms [
Therefore, understanding the link between the MAPK and PI3K signaling pathways becomes important. It is known that autophosphorylation of the insulin receptor tyrosine kinase occurs after combining with insulin and then activates IRS-1. Subsequently the tyrosine (Tyr) residues of IRS-1 are phosphorylated and serve to recruit proteins containing the Src homology 2 domain, including class I-PI3K molecules; hence, the phosphorylation of IRS-1 on the tyrosine residues is the important prerequisite for the activation of downstream signaling pathways. In type 2 DM, the excessive phosphorylation of serine (Ser) residues of IRS-1 inhibits the phosphorylation of the Tyr residues [
However, not all of the Ser residues of IRS-1 have an inhibitory effect on insulin signaling. Some studies have suggested that certain Ser residues can inhibit Tyr phosphorylation of IRS-1 due to their proximity to phosphotyrosine-binding domains (PTB) (e.g., murine Ser302, Ser307; human Ser307, Ser312) or PI3K binding site (e.g., murine Ser612; human Ser616). It has been found that when Ser302, Ser307, and Ser612 of IRS-1 were changed to alanine (Ala) by point mutation in mice at the same time, phosphorylation at these three sites was blocked; the transgenic mice showed an improved glucose tolerance and insulin sensitivity, which was accompanied by elevated phosphorylation levels of the Tyr residue of IRS-1 and PI3K [
In conclusion, the present study evaluated the therapeutic effect of QDTS granules on DM kidney injury and insulin resistance, revealing that QDTS granules could restore the phosphorylation balance between the Ser and Tyr residues of IRS-1 via inhibiting the activation of MAPK pathway. The current views hold that the positive or negative feedback mechanism or the concept of a regulatory network in molecular biology is very similar to the concept of “yin and yang" used in the theory of TCM [
The data sets used and analyzed during the current study are available from the corresponding author on reasonable request.
The authors declare that they have no conflicts of interest.
Hongfang Liu designed the study and coordinated the experiments and draft of the manuscript. Xue Gao carried out the animal study and participated in the experimental design and data analyses and completed the manuscript. Zhichao An and Qiying He were involved in the animal study, immunohistochemical staining, and western blot. All authors read and approved the final manuscript.
The authors are grateful to all study participants. They also thank Zhuozhou Dongle Pharmaceutical Co. for providing the water extract of the prescription. This work was supported by Beijing Municipal Natural Science Foundation, Project no. 7162123, and Beijing Municipal Science and Technology Commission, Project no. Z161100001816003.