Huaju Xiaoji Formula Regulates ERS-lncMGC/miRNA to Enhance the Renal Function of Hypertensive Diabetic Mice with Nephropathy

Abstract

Background: Better therapeutic drugs are required for treating hypertensive diabetic nephropathy. In our previous study, the Huaju Xiaoji (HJXJ) formula promoted the renal function of patients with diabetes and hypertensive nephropathy. In this study, we investigated the therapeutic effect and regulation mechanism of HJXJ in hypertensive diabetic mice with nephropathy.

Methods: We constructed a mouse hypertensive diabetic nephropathy (HDN) model by treating mice with streptozotocin (STZ) and nomega-nitro-L-arginine methyl ester (LNAME). We also constructed a human glomerular mesangial cell (HGMC) model that was induced by high doses of sugar (30 mmol/mL) and TGFβ1 (5 ng/mL). Pathological changes were evaluated by hematoxylin and eosin (H&E) staining, periodic acid Schiff (PAS) staining, and Masson staining. The fibrosis-related molecules (TGFβ1, fibronectin, laminin, COL I, COL IV, α-SMA, and p-smad2/3) were detected by enzyme-linked immunosorbent assay (ELISA). The mRNA levels and protein expression of endoplasmic reticulum stress, fibrosis molecules, and their downstream molecules were assessed using qPCR and Western blotting assays.

Results: Administering HJXJ promoted the renal function of HDN mice. HJXJ reduced the expression of ER stress makers (CHOP and GRP78) and lncMGC, miR379, miR494, miR495, miR377, CUGBP2, CPEB4, EDEM3, and ATF3 in HDN mice and model HGMCs. The positive control drugs (dapagliflozin and valsartan) also showed similar effects after treatment with HJXJ. Additionally, in model HGMCs, the overexpression of CHOP or lncMGC decreased the effects of HJXJ-M on the level of fibrosis molecules and downstream target molecules.

Conclusion: In this study, we showed that the HJXJ formula may regulate ERS-lncMGC/miRNA to enhance renal function in hypertensive diabetic mice with nephropathy. This study may act as a reference for further investigating whether combining HJXJ with other drugs can enhance its therapeutic effect. The findings of this study might provide new insights into the clinical treatment of hypertensive diabetic nephropathy with HJXJ.

Figure 1

HJXJ intervention improved renal function in HDN mice after 12 weeks. (a) Urinary albumin. (b) Serum creatinine. (c) Blood urea nitrogen. (d) Blood triglycerides. (e) Total cholesterol. (f) High-density lipoprotein (HDL) cholesterol. (g) Low-density lipoprotein (LDL)/very-low-density lipoprotein (VLDL) cholesterol. ^∗∗p < 0.01, vs. Con group; ^#p < 0.05 and ^##p < 0.01, vs. STZ-LNAME group.

Figure 2

HJXJ intervention ameliorated the pathological changes in HDN mice after 12 weeks. Representative images of kidney tissues stained with H&E; tissue sections were observed at 200x magnification (scale bar = 100 μm) and 400x magnification (scale bar = 50 μm). Black arrowheads indicate glomerulus. Blue arrowheads indicate Bowman capsules. Red arrowheads indicate glomerular space. Yellow asterisks indicate interstitial regions. Representative images of kidney tissues stained with PAS; tissue sections were observed at 200x magnification (scale bar = 100 μm) and 400x magnification (scale bar = 50 μm). Representative images of kidney tissues were stained with Masson; tissue sections were observed at 200x magnification (scale bar = 100 μm) and 400x magnification (scale bar = 50 μm).

Figure 3

HJXJ intervention reduced fibrosis in STZ-LNAME-induced mice after 12 weeks. (a) ELISA was performed to detect the level of TGFβ1, fibronectin, and laminin in the serum of HDN mice after 12 weeks of HJXJ intervention. (b) ELISA was performed to detect the level of TGFβ1, fibronectin, laminin, and collagen (COL I and COL IV) in the serum of HDN mice after 12 weeks of HJXJ intervention. (c) The mRNA level of α-SMA in the kidney tissue of HDN mice after 12 weeks of HJXJ intervention was evaluated by RT-qPCR. (d) Western blotting assays were performed to measure the level of α-SMA, Smad2/3, and p-smad2/3 in the kidney tissue of HDN mice after 12 weeks of HJXJ intervention. ^∗∗p < 0.01, vs. Con group; ^#p < 0.05 and ^##p < 0.01, vs. STZ-LNAME group.

Figure 4

HJXJ intervention decreased ER stress and the expression of the downstream molecules after STZ-LNAME induction. (a) The mRNA levels of the ER stress chaperone genes (CHOP and GRP78) were detected by RT-qPCR. (b) The protein levels of CHOP and GRP78 were estimated by Western blotting assays. (c) RT-qPCR analysis was performed to detect the expression of ER stress-induced lncMGC, miR379, miR494, miR495, and miR377. (d) The mRNA levels of CUGBP2, CPEB4, EDEM3, and ATF3 were detected by RT-qPCR analysis. (e) The protein levels of CUGBP2, CPEB4, EDEM3, and ATF3 were estimated by Western blotting assays. (f) The histogram analysis of the grey intensity of CUGBP2, CPEB4, EDEM3, and ATF3. ^∗∗p < 0.01, vs. Con group; ^#p < 0.05 and ^##p < 0.01, vs. STZ-LNAME group.

Figure 5

HJXJ treatment decreased the level of fibrosis molecules in HGMCs (model HGMC) induced by high doses of sugar (30 mmol/mL) and TGFβ1 (5 ng/mL). (a) ELISA was performed to assess the level of fibronectin and laminin. (b) ELISA was performed to assess the collagen (COL I and COL IV) content. (c) The mRNA level of α-SMA was evaluated by RT-qPCR analysis. (d) Western blotting assays were performed to measure the levels of α-SMA, Smad2/3, and p-smad2/3. ^∗∗p < 0.01, vs. Con group; ^#p < 0.05 and ^##p < 0.01, vs. sugar+TGFβ1 group.

Figure 6

HJXJ intervention decreased ER stress and the expression of the downstream molecules in HGMCs induced by high doses of sugar (30 mmol/mL) and TGFβ1 (5 ng/mL). (a) The mRNA levels of ER stress chaperone genes (CHOP and GRP78) were detected by RT-qPCR analysis. (b) The protein levels of CHOP and GRP78 were measured by Western blotting assays. (c) RT-qPCR was performed to evaluate the expression of ER stress-induced lncMGC, miR379, miR494, miR495, and miR377. (d) The mRNA levels of CUGBP2, CPEB4, EDEM3, and ATF3 were detected by RT-qPCR. (e) The protein levels of CUGBP2, CPEB4, EDEM3, and ATF3 were estimated by Western blotting assays. (f) The histogram analysis of the grey intensity of CUGBP2, CPEB4, EDEM3, and ATF3 was performed. ^∗∗p < 0.01, vs. Con group; ^#p < 0.05 and ^##p < 0.01, vs. the STZ-L-NAME group.

Figure 7

The overexpression of CHOP or lncMGC decreased the effect of HJXJ-M on fibrosis in HGMCs induced by high doses of sugar (30 mmol/mL) and TGFβ1 (5 ng/mL). (a) ELISA was performed to detect the levels of fibronectin and laminin in HGMCs. (b) ELISA was performed to determine the level of collagen (Col I and Col IV) in HGMCs. (c) The mRNA level of α-SMA in HGMCs was evaluated by RT-qPCR. (d) Western blotting analysis was performed to estimate the level of α-SMA, Smad2/3, and p-smad2/3 in HGMCs. (e) The histogram analysis of the grey intensity of α-SMA, Smad2/3, and p-smad2/3 in HGMCs. ^∗∗p < 0.01, vs. Con group; ^#p < 0.05 and ^##p < 0.01, vs. sugar+TGFβ1 group; ^$p < 0.05 and ^$$p < 0.01, vs. HJXJ group.

Figure 8

The overexpression of CHOP or lncMGC regulated the effects of HJXJ on the levels of lncMGC, miR379, miR494, miR495, and miR377 in HGMCs after treatment with high doses of sugar (30 mmol/mL) and TGFβ1 (5 ng/mL). RT-qPCR was performed to detect the expression of ER stress-induced lncMGC, miR379, miR494, miR495, and miR377. ^∗∗p < 0.01, vs. Con group; ^##p < 0.01, vs. sugar+TGFβ1 group; ^$$p < 0.01, vs. HJXJ group.

Figure 9

The overexpression of CHOP or lncMGC regulated the effects of HJXJ on the expression of CUGBP2, CPEB4, EDEM3, and ATF3 in HGMCs after treatment with high doses of sugar (30 mmol/mL) and TGFβ1 (5 ng/mL). (a) The mRNA levels of CUGBP2, CPEB4, EDEM3, and ATF3 were detected by RT-qPCR. (b) The protein levels of CUGBP2, CPEB4, EDEM3, and ATF3 were measured by Western blotting assays. ^∗∗p < 0.01, vs. Con group; ^##p < 0.01, vs. sugar+TGFβ1 group; ^$$p < 0.01, vs. HJXJ group.

Figure 10

The Huaju Xiaoji formula influences ERS-lncMGC/miRNA to regulate the extracellular matrix proteins in hypertensive diabetic nephropathy.