Effect of phosphatase and tensin homolog-induced putative kinase 1/ E3 ubiquitin ligase Parkin mediated mitochondrial autophagy on chronic kidney disease myocardial injury and the intervention mechanism of Shenshuai recipe (肾衰方)

doi:10.19852/j.cnki.jtcm.20231231.001

Abstract

Abstract:

OBJECTIVE: To study whether Shenshuai recipe (肾衰方, SSR) can play a protective role on chronic kidney disease myocardial injury model through phosphatase and tensin homolog-induced putative kinase 1 (PINK1)/E3 ubiquitin ligase Parkin (Parkin) mitochondrial autophagy pathway.

METHODS: Forty-eight nephrectomized rats were randomly divided into six groups: sham-operated group, model group, Benazepril group, low, medium and high-dose groups of SSR. The rats were given the cor-responding intervention for six weeks, then were sacrificed. Serum was examined by enzyme linked immunosorbent assay (ELISA). Cardiac ultrasound was used to detect cardiac function in 5/6 nephrectomized rats. Myocardial tissue was examined by light and electron microscopy; PINK1, Parkin, microtubule-associated protein1 light chain 3 II (LC3B), sequestosome 1 (P62), BECN1 (Beclin-1) and dynamin-related protein 1 (Drp-1) were measured by real time polymerase chain reaction (RT-PCR), Western blot (WB) and immunohistochemistry (IHC).

RESULTS: The expression levels of blood urea nitrogen (BUN) and creatinine (SCr) in the model group were significantly higher than those in the sham-operated group, indicating that modeling was successful. SSR can protect myocardium by reducing the relative expression of creatine kinase myocardial isoenzyme and hypersensitivity cardiac troponin I (P<0.05). SSR can improve cardiac function in rats after ultrasound testing. SSR can improve the pathological manifestations of myocardial tissue after Masson staining. SSR can increase the number of autophagosomes and autophagiclysosomes in 5/6 nephrectomized rats (P<0.05). Determined by RT-PCR, WB and IHC, SSR can increase the relative expression of PINK1, Parkin, and LC3B (P<0.05), and decrease the relative expression of P62, Beclin-1 and Drp-1 (P<0.05).

CONCLUSIONS: The PINK1/Parkin mitochondrial autophagy pathway in myocardial tissues in 5/6 nephrectomy CKD myocardial injury rats was inhibited. SSR can activate PINK1/Parkin mitochondrial autophagy to enhance mitochondrial autophagy, and play a protective role in myocardial tissues.

Key words: renal insufficiency, chronic, PTEN phosphohydrolase, ubiquitin-protein ligases, myocardial injury, mitochondrial autophagy, Shenshuai recipe

ZHANG Gedi, LIU Gengxin, GUO Min, LUO Fuli, YAN Ziyou, GE Wei. Effect of phosphatase and tensin homolog-induced putative kinase 1/ E3 ubiquitin ligase Parkin mediated mitochondrial autophagy on chronic kidney disease myocardial injury and the intervention mechanism of Shenshuai recipe (肾衰方)[J]. Journal of Traditional Chinese Medicine, 2024, 44(5): 934-943.

Figures/Tables 11

Table 1 Comparison of the relative expression of BUN and SCr in serum of rats in each group ($\bar{x}±s$)

Group	n	BUN	SCr
Sham operated	6	8.1±0.6	56.9±12.1
Model	5	38.2±11.8^a	122.7±28.9^a
Benazepril	6	36.1±11.3	118.8±28.5
Low dose	6	31.2±9.8	102.2±28.7
Medium dose	6	29.1±10.3	98.1±26.7
High dose	6	27.3±10.0	93.8±27.8

Table 2 Comparison of the relative expression of CM-MB and hs-cTnI in serum of rats in each group (ratio, $\bar{x}±s$)

Group	n	CK-MB	hs-cTnI
Sham operated	6	1.00±0.14	1.00±0.08
Model	5	1.47±0.13^a	1.61±0.17^a
Benazepril	6	1.13±0.05^b	1.06±0.18^b
Low dose	6	1.09±0.11^b	1.02±0.14^b
Medium dose	6	1.05±0.11^b	1.16±0.11^b
High dose	6	1.11±0.13^b	1.31±0.07^b

Table 3 Comparison of the cardiac function in each group ($\bar{x}±s$)

Group	n	EF%	FS%	LVEDD (mm)	LVESD (mm)
Sham-operated	6	80.1±10.6	56.9±13.1	7.8±2.0	5.2±1.1
Model	5	38.2±3.8^a	30.7±5.9^a	23.5±7.7^a	6.1±1.3
Benazepril	6	49.1±4.3^b	39.8±8.5	13.9±3.6^b	5.5±1.2
Low dose	6	43.2±2.8	33.2±7.7	18.1±4.9	5.9±1.1
Medium dose	6	48.1±3.3^b	38.1±6.7	16.6±4.6	5.1±1.0
High dose	6	50.3±4.3^b	40.8±6.8	14.8±3.1^b	5.3±1.1

Figure 1 Pathology of rats in each group after HE staining A: sham-operated group (× 40); B: Model group (× 40); C: Benazepril group (× 40); D: Low dose group (× 40); E: Meduim dose group (× 40); F: High dose group (× 40)；G: sham-operated group (× 200); H: Model group (× 200); I: Benazepril group (× 200); J: Low dose group (× 200); K: Meduim dose group (× 200); L: High dose group (× 200). Sham-operated groups and Model group were given normal saline 2 mL per day for 6 weeks; Benazepril group was given atorvastatin at 1.5 mg·kg-1·d-1 by gavage for 6 weeks; Low dose group (9.75 g·kg-1·d-1), Medium dose group (19.5 g·kg-1·d-1) and High dose group (39 g·kg-1·d-1) were given the corresponding dose by gavage for 6 weeks. Black arrow: myocardial fibers are loose and broken; Blue arrow：fibrous stroma is widened.

Figure 2 Autophagosomes and autophagiclysosomes of rats in each group under electron microscope A: sham-operated group (× 7000); B: Model group (× 7000); C: Benazepril group (× 7000); D: Low dose group (× 7000); E: Meduim dose group (× 7000); F: High dose group (× 7000). Sham-operated groups and Model group were given normal saline 2 mL per day for 6 weeks; Benazepril group was given atorvastatin at 1.5 mg·kg-1·d-1 by gavage for 6 weeks; Low dose group (9.75 g·kg-1·d-1), Medium dose group (19.5 g·kg-1·d-1) and High dose group (39 g·kg-1·d-1) were given the corresponding dose by gavage for 6 weeks. Red arrow: autophagosomes; Blue arrow: autophagiclysosomes.

Table 4 Comparison of the number of autophagic bodies and autophagiclysosomes of rats in each group ($\bar{x}±s$)

Group	n	Autophagosome	Autophagolysosome
Sham-operated	6	13.6±4.9	3.0±1.0
Model	5	11.0±3.0	3.0±2.0
Benazepril	6	23.0±4.3^a	12.6±3.2^a
Low dose	6	14.3±7.2	10.6±1.1^a
Medium dose	6	24.6±1.53^a	18.0±3.0^a
High dose	6	31.3±5.0^a	16.6±2.3^a

Figure 3 Western blot strips of proteins in each group 1 : Sham-operated group, 2; Model group, 3: Benazepril group, 4: Low dose group, 5: Medium group, 6; High dose group. Sham-operated groups and Model group were given normal saline 2 mL per day for 6 weeks; Benazepril group was given atorvastatin at 1.5 mg·kg-1·d-1 by gavage for 6 weeks; Low dose group (9.75 g·kg-1·d-1), Medium dose group (19.5 g·kg-1·d-1) and High dose group (39 g·kg-1·d-1) were given the corresponding dose by gavage for 6 weeks.

Figure 4 Immunohistochemical expression pictures of PINK1, Parkin, LC3B and P62 A: PINK1, B: Parkin, C: LC3B, D: P62. A1, B1, C1, D1: Sham-operated group; A2, B2, C2, D2: Model group; A3, B3, C3, D3: Benazepril group; A4, B4, C4, D4: Low dose group; A5, B5, C5, D5: Medium group; A6, B6, C6, D6: High dose group. Sham-operated groups and Model group were given normal saline 2 mL per day for 6 weeks; Benazepril group was given atorvastatin at 1.5 mg·kg-1·d-1 by gavage for 6 weeks; Low dose group (9.75 g·kg-1·d-1), Medium dose group (19.5 g·kg-1·d-1) and High dose group (39 g·kg-1·d-1) were given the corresponding dose by gavage for 6 weeks. PINK1: phosphatase and tensin homolog-induced putative kinase 1; Parkin: E3 ubiquitin ligase Parkin; LC3B: microtubule-associated protein1 light chain 3 Ⅱ; P62: sequestosome 1.

Table 5 Comparison of the relative expression of PINK1, Parkin, LC3B, P62, Beclin-1, and Drp-1 mRNA in myocardial tissue of rats in each group ($\bar{x}±s$)

Group	n	PINK1	Parkin	LC3B	P62	Beclin-1	Drp-1
Sham-operated	6	1.04±0.33	1.11±0.22	1.11±0.20	0.86±0.15	0.76±0.09	1.00±0.23
Model	5	0.58±0.08^a	0.41±0.23^a	0.19±0.04^a	1.49±0.43^a	1.33±0.32^a	1.61±0.08^a
Benazepril	6	0.99±0.05^b	1.00±0.35^b	0.70±0.05^b	0.59±0.10^b	0.52±0.11^b	0.84±0.05^b
Low dose	6	1.00±0.17^b	1.30±0.41^b	0.57±0.24^b	0.64±0.04^b	0.67±0.08^b	1.34±0.04^b
Medium dose	6	0.98±0.13^b	1.40±0.41^b	0.70±0.05^b	0.62±0.16^b	0.22±0.06^b	1.02±0.22^b
High dose	6	1.06±0.24^b	1.17±0.43^b	0.84±0.10^b	0.24±0.09^b	0.74±0.17^b	1.07±0.16^b

Table 6 Comparison of the relative expression of PINK1, Parkin, LC3B, P62, Beclin-1, and Drp-1 protein in myocardial tissue of rats in each group (WB, $\bar{x}±s$)

Group	n	PINK1	Parkin	LC3B	P62	Beclin-1	Drp-1
Sham-operated	6	0.66±0.12	0.91±0.16	0.99±0.15	0.72±0.14	0.96±0.19	0.62±0.06
Model	5	0.40±0.05^a	0.51±0.01^a	0.42±0.06^a	1.13±0.20^a	1.32±0.22^a	1.04±0.25^a
Benazepril	6	0.89±0.12^b	0.78±0.04^b	0.77±0.09^b	0.68±0.14^b	0.72±0.19^b	0.50±0.27^b
Low dose	6	0.66±0.15^b	0.81±0.07^b	0.67±0.19^b	0.54±0.15^b	0.88±0.22^b	0.58±0.05^b
Medium dose group	6	0.68±0.11^b	0.86±0.08^b	0.76±0.10^b	0.79±0.11^b	0.60±0.11^bb	0.51±0.23^b
High dose	6	0.64±0.11^b	0.85±0.09^b	0.92±0.12^b	0.39±0.10^b	0.89±0.16^bb	0.34±0.24^b

Table 7 Comparison of the area value of PINK1, Parkin, LC3B and P62 protein in myocardial tissue of rats in each group (IHC,$\bar{x}±s$)

Group	n	PINK1	Parkin	LC3B	P62
Sham-operated	6	183079±20650	265533±26591	267318±8622	38781±5733
Model	5	70026±11298^a	91809±14756^a	67988±10397^a	71151±13587^a
Benazepril	6	113379±30689^b	161869±23557^b	160131±27944^b	46035±10737^b
Low dose	6	102011±139^b	141255±13478^b	120706±14396^b	40491±5294^b
Medium dose	6	127373±18125^b	152109±20738^b	163192±20119^b	32060±9663^b
High dose	6	140242±22569^b	177205±20136^b	188348±12890^b	21825±7940^b

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