Dendrobium mixture improved diabetic nephropathy in db/db 1 mice by regulating TGF-β1/Smads signal transduction

9 Background: Dendrobium mixture (DMix) is an effective treatment for diabetic nephropathy 10 (DN), but the underlying molecular mechanism remains unclear. In this study, we 11 investigated whether DMix regulates the transforming growth factor-β1 (TGF-β1)/Smads 12 signal transduction pathway. 13 Methods: Twenty-four db/db mice were randomly divided into three groups: the model, 14 DMix, and gliquidone groups, while eight db/m mice were selected as the normal control 15 group. The drug was administered by continuous gavage for 8 weeks. Body weight (BW), 16 kidney weight (KW), kidney index, fasting blood glucose (FBG), blood lipid, 24-hour urinary albumin excretion rate, blood urea nitrogen, and serum creatinine levels were measured. Pathological changes in the renal tissue were observed using a light microscope. Real-time 19 quantitative PCR and immunohistochemical staining were used to detect mRNA expression 20 of TGF-β1 and alpha-smooth muscle actin (α-SMA) genes and proteins, respectively, in renal 21 tissues. TGF-β1, Smad2, p-Smad2, Smad3, p-Smad3, and α-SMA expression levels were 22 measured using western blotting. 23 Results: DMix significantly reduced FBG level, BW, KW, and blood lipid level, and 24 improved renal function in db/db mice. Histopathology showed that DMix alleviated 25 glomerular mesangial cell proliferation and renal interstitial fibrosis in db/db mice. 26 Additionally, DMix reduced protein and mRNA expression of TGF-β1 and α-SMA, and 27 inhibited Smad2 and Smad3 phosphorylation. 28 Conclusions: The findings suggest that DMix may inhibit renal fibrosis and delay the 29 progression of DN by regulating the TGF-β1/Smads signaling pathway. 30


33
Diabetic nephropathy (DN) is a common chronic microvascular complication of diabetes and 34 the most important cause of death in patients with diabetes [1,2]. DN is characterized by the 35 thickening of the glomerular basement membrane, proliferation of mesangial cells, and 36 accumulation of extracellular matrix, leading to glomerulosclerosis and interstitial fibrosis 37 [3,4]. Transforming growth factor-β1 (TGF-β1) is a key cytokine-promoting fibrosis, and the 38 Smad protein is the intracellular kinase substrate of the TGF-β1 receptor, mediating the TGF-39 6 fiber showed a reddish color and was soaked in 2% aniline blue solution for 5 min. Then, 107 dehydration and transparent sealing were performed before observation under a light 108 microscope. 109

Real-time quantitative PCR (RT-qPCR) 110
Total RNA was extracted from mice kidney tissue with RNAiso Plus reagent (Takara, Tokyo, 111 Japan), and the concentration was determined. Then, cDNA was synthesized by reverse 112 transcription using a reverse transcription kit (Takara, Tokyo, Japan). The PCR reaction was 113 performed using a PCR kit (Takara, Tokyo, Japan) under the following reaction conditions: 114 denaturation, 95 °C for 30 s; annealing, 55 °C for 30 s; extension, 72 °C for 1 min; 30 cycles. 115 SDS 2.4 software was used to analyze the CT values of the samples detected during the PCR 116 process, using β-actin as the internal referenceand adopting the ΔΔCt method for relative 117 quantitative analysis, with 2 -△△ Ct as a quantity relative expression of the target RNA. PCR 118 primers (Table 1)  The kidney tissue was fixed in 4% paraformaldehyde solution, embedded in paraffin, cut into 124 4-μm-thick slices, baked for 2 h, dewaxed using xylene twice, hydrated with gradient alcohol, 125 placedinto boiled sodium citrate solution for antigen repair, and cooled naturally to room 126 temperature (18-30 °C). The sections were rinsed with phosphate-buffered saline (PBS) 127 thrice, co-incubated with an endogenous peroxidase blocker at room temperature for 10 min, 128 rinsed with PBS thrice, and co-incubated with non-immunized animal serum at room 129 temperature for 10 min. After removing the serum, primary antibodies were added drop-wise 130 as follows: rabbit anti-TGF-β1 and anti-α-SMA polyclonal antibodies (1:100 dilution each, 131 Abcam, Cambridge, UK), incubated at 4 °C overnight, and rinsed with PBS thrice; biotin-132 labeled sheep anti-rabbit IgG (ready to use, Fuzhou Maixin BiotechCo., Ltd, Fuzhou, China), 133 incubated at room temperature for 10 min, and rinsed with PBS thrice; streptavidin-134 peroxidase (Fuzhou Maixin BiotechCo., Ltd, Fuzhou, China), incubated at room temperature 135 for 10 min, and rinsed with PBS thrice. Then, DAB (Wuhan Boster Biological Technology  136 Co., Ltd, Wuhan, China) was added for color development, rinsed with distilled water, 137 hematoxylin-dyed, and tap water-rinsed for blueness. The gradient alcohol was dehydrated 138 and dried, xylene was transparent, neutral gum was sealed, and tan was positively expressed 139 under the optical microscope. The Image-pro Plus 6.0 Image analysis software was used for 140 semi-quantitative analysis, and the relative protein expression was represented by the mean 141 density. 142

Western blot assays 143
Kidney tissues stored in liquid nitrogen mixed with appropriate protein lysate were fully 144 ground to produce tissue homogenate. After centrifugation (4 °C, 12,000 rpm, 15 min), the 145 total protein was extracted from the supernatant and the protein concentration was 146 8 determined using the bicinchoninic acid assay. Then, 30μg of each sample was used for 10% 147 SDS-PAGE gel electrophoresis, transferred to a polyvinylidene fluoride membrane, and 148 sealed with 5% skim milk at room temperature for 1 h. Primary antibodies (TGF-β1, Smad2, 149 p-Smad2, Smad3, p-Smad3, α-SMA) were added and incubated with the membrane overnight 150 at 4 °C. After rinsing with tris-buffered saline, 0.1% Tween 20 (TBST), the membrane was 151 incubated with the secondary antibody at room temperature for 1 h. After TBST rinsing, the 152 membrane was stained using enhanced chemiluminescence and viewed using a gel imaging

Statistical analyses 163
SPSS 22.0 statistical software was used to analyze the data, which are expressed as mean ± 164 standard deviation (SD). Differences among multiple sample groups were analyzed using 165 one-way ANOVA. The Bonferroni method was used for pairwise comparison between 166 groups when the variances were homogeneous, and Tamhane's T2 comparison was used 167 when the variances were heterogeneous. P<0.05 was considered statistically significant. 168

Comparison of general signs 170
Mice in the normal group were in a good mental state, responsive, with shiny hair, and in a 171 good feeding condition. db/db Mice were listless and unresponsive, with increased diet and 172 urine volumes; the above symptoms of mice in each treatment group were improved to 173 different degrees compared with the model group. 174

DMix reduced FBG levels of db/db mice 175
The FBG level in db/db mice was approximately 3× higher than that in the normal group 176 The serum TC and TG levels of mice in the model group were significantly higher than those 202 in the normal group (P<0.01). TC and TG levels in both the Dmix and gliquidone groups 203 were significantly lower than those in the model group (TG, P<0.05; TC, P<0.01) (Fig. 3a, b). 204 There was no significant difference between the Dmix and gliquidone groups (P>0.05). 205 These results indicate that DMix could regulate lipid metabolism. DMix group were significantly lower than those in the model group (P<0.05), but there was 12 no significant difference between the DMix and gliquidone groups (P>0.05) (Fig. 4a-c). 218 These results indicate that DMix had a protective effect on the kidney of db/db mice. The activation of the Smad pathway and its subsequent nuclear transposition are key steps in 287 TGF-β1-mediated renal fibrosis in DN [13]. The phosphorylation of Smad2 and Smad3 is 288 also an important signal transduction process in the TGF-β1/Smads signaling pathway, and 289 their expression indicates TGF-β1/Smads signaling pathway activation [14]. The expression 290 of TGF-β1, Smad2, p-Smad2, Smad3, p-Smad3, and α-SMA in mouse renal tissues was 291 measured via western blotting. The protein expression of TGF-β1, p-Smad2, p-Smad3, and α-292 SMA in the model group was significantly higher than that in the normal group (P<0.01), 293 indicating that the TGF-β1/Smads signaling pathway was activated in db/db mouse renal 294 tissue. After 8 weeks of treatment with DMix, the expression of TGF-β1, p-Smad2, p-Smad3, 295 and α-SMA proteins was significantly lower than that in the model group (TGF-β1:β-actin, p-296 Smad2:Smad2, and α-SMA:β-actin, P<0.05; p-Smad3:Smad3, P<0.01), but there was no 297 significant change in the expression of the Smad2 and Smad3 proteins (Fig. 8a-d). There was 298 no significant difference between the DMix and gliquidone groups (P>0.05). Western blots 299 show that DMix inhibited the TGF-β1/Smads signaling pathway in the renal tissues of db/db 300 mice. 301 of this experiment showed that db/db mice had a significantly greater body weight than db/m 321 mice. Additionally, blood glucose, Scr and BUN levels, and KI were significantly higher in 322 db/db mice than in the normal group. Furthermore, the db/db mice exhibited proteinuria, 323 dyslipidemia, glomerular hypertrophy, and fibrosis, confirming that the DN model was 324 successful. After treatment with DMix, these parameters were significantly attenuated (Fig.  325   1-4), which was consistent with previous studies and our clinical observation [9][10][11]. 326 Additionally, HE, PAS, and Masson staining showed that the degree of renal pathological 327 injury and fibrous hyperplasia improved significantly in the model group with the 328 administration of DMix (Fig. 5). DN is characterized by proteinuria and glomerular sclerosis 329 20 [23,24], and our results indicate that DMix not only reduces urinary protein levels, but also 330 reduces renal fibrosis, suggesting that DMix effectively prevents the development of DN. 331 The pathogenesis of DN is complex and has not been fully elucidated. Renal interstitial 332 fibrosis is an important mechanism of renal deterioration in the pathogenesis of DN. 333 Therefore, the key to delay the development of DN is to inhibit renal interstitial fibrosis 334 [25,26]. TGF-β1/Smads is the core pathway of renal fibrosis and one of the important factors 335 in the development of DN [27,28]. TGF-β1 is considered an important factor contributing to 336 renal mesenchymal fibrosis, and previous studies have confirmed that TGF-β1 is over 337