Neuroprotective effect of Naochuxue prescription (脑出血方) on intracerebral hemorrhage: inhibition of autophagy via downregulating high mobility group box-1

doi:10.19852/j.cnki.jtcm.20240515.003

Abstract

Abstract:

OBJECTIVE: To determine the molecular mechanisms underlying the neuroprotective effects of Naochuxue prescription (脑出血方,NCXP) in rats with intracerebral hemorrhage (ICH).

METHODS: Sprague-Dawley rats were injected with collagenase to generate ICH models, which were then randomly divided into six groups, including control, sham, model, and three intervention groups. The intervention groups received different doses of NCXP (0.13, 0.26, and 0.52 g/kg) daily for 10 d. High-performance liquid chromatography (HPLC) was used to analyze the chemical characteristics of NCXP. The neurobehavioral outcomes of the rats were evaluated using neurological deficit scores (Zea Longa 5) and the corner turn test. Pathomorphological changes in perihematomal tissues after ICH were observed using hematoxylin and eosin staining. Immunohistochemistry (IHC) was used to detect the inflammation expression of interleukin 6 (IL-6) and toll-like receptor 4 (TLR4). High mobility group box-1 (HMGB1), Beclin1, microtubule-associated protein 1 light chain 3 beta (LC3), and sequestosome 1 (p62) were detected using real-time quantitative polymerase chain reaction and Western blotting in perihematomal tissues.

RESULTS: HPLC showed that the NCXP had good stability. Rats with ICH had severe neurological function deficits compared to the control group. IHC results showed that NCXP significantly downregulated the expression of the inflammatory proteins IL-6 and TLR4. ICH rats treated with NCXP showed less neurological injury than the model group, accompanied by a significantly decreased expression of HMGB1, Beclin1, and LC3 and an increased expression of p62.

CONCLUSIONS: The neuroprotective effect of NCXP alleviated inflammation and autophagy possibly by downregulating HMGB1 expression. However, further research on the signaling pathways is required to verify this hypothesis.

Key words: intracerebral hemorrhage, HMGB proteins, inflammation, autophagy, Naochuxue prescription

JIN Hong, WANG Xinna, WANG Ruonan, LI Jinjian, YU Junchao, ZHAO Dexi, ZHAI Lu. Neuroprotective effect of Naochuxue prescription (脑出血方) on intracerebral hemorrhage: inhibition of autophagy via downregulating high mobility group box-1[J]. Journal of Traditional Chinese Medicine, 2024, 44(5): 944-953.

Figures/Tables 4

Figure 1 HPLC chromatogram of NCXP A: HPLC mixed standard chromatograms of Typhaneoside, Isorhamnetin-3-O-neohespeidoside, Aloe-Emodin, Rhein, Emodin, and Chrysophanol with DAD detection at 254 nm are shown. B: HPLC chromatogram of NCXP with DAD detection at 254 nm is shown. C: HPLC fingerprint chromatogram of ten batches of NCXP (S1-10) and similarity was analyzed using the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition). HPLC: high-performance liquid chromatography; NCXP: Naochuxue prescription; DAD: diode array detector.

Figure 2 Effects of NCXP on neurobehavioral outcomes after ICH A: neurological deficit scores (Zea Longa 5) on the 1st, 3rd, 5th, and 10th days after ICH; B: corner turn test on the 1st, 3rd, 5th, and 10th days after ICH. Control: control group; Sham: sham-operated group; Model: ICH model group; NCXP (L): low-dose Naochuxue prescription (0.13 g/kg); NCXP (M): medium-dose Naochuxue prescription (0.26 g/kg); NCXP (H): high-dose Naochuxue prescription (0.52 g/kg). The drug was administered by once daily for 10 d. NCXP: Naochuxue prescription; ICH: intracerebral hemorrhage. One-way analysis of variance, and Tukey's multiple comparisons test. Results are presented as mean ± standard deviation. aP<0.01, vs sham group; bP<0.05,cP<0.01, vs model group; dP<0.01, vs control group, n = 6 in each group.

Figure 3 Pathological effects and the expression of IL-6 and TLR4 of NCXP on the brain tissue after ICH A: pathological effects of each group tissue (× 200). The dyeing method of the pictures is the hematoxylin-eosin staining method. A1: control group; A2: sham group; A3: model group; A4: NCXP (L) group; A5: NCXP (M) group; A6: NCXP (H) group; B: effect of different doses of NCXP on the expression of IL-6 (× 400). B1: control group; B2: sham group; B3: model group; B4: NCXP (L) group; B5: NCXP (M) group; B6: NCXP (H) group; C: effect of different doses of NCXP on the expression of TLR4 (× 400). C1: control group; C2: sham group; C3: model group; C4: NCXP (L) group; C5: NCXP (M) group; C6: NCXP (H) group. The Dyeing method of the Figures B-C is the immunohistochemistry method; D: statistical graph of IL-6 expression; E: statistical graph of TLR4 expression. Control: control group; Sham: sham-operated group; Model: ICH model group; NCXP (L): low-dose Naochuxue prescription (0.13 g/kg); NCXP (M): medium-dose Naochuxue prescription (0.26 g/kg); NCXP (H): high-dose Naochuxue prescription (0.52 g/kg). The drug was administered by once daily for 10 d. NCXP: Naochuxue prescription; ICH: intracerebral hemorrhage; IL-6: interleukin 6; TLR4: toll-like receptor 4. One-way analysis of variance, and Tukey's multiple comparisons test. Results are presented as mean ± standard deviation. aP>0.05, bP<0.01, vs Control; cP<0.01 vs Model, n = 6 in each group.

Figure 4 mRNA and protein expression of HMGB1, Beclin1, LC3, and p62 of NCXP on the brain tissue after ICH A: mRNA expression of HMGB1, Beclin1, LC3B, and p62; A1: mRNA expression of HMGB1, A2: mRNA expression of Beclin1, A3: mRNA expression of LC3B, A4: mRNA expression of p62. B: protein images and expression of HMGB1, Beclin1, LC3Ⅱ/Ⅰ, and p62. B1: Western blotting representative images of HMGB1, Beclin1, LC3Ⅱ/Ⅰ, and p62, B2: protein expression of HMGB1, B3: protein expression of Beclin1, B4: protein expression of LC3Ⅱ/Ⅰ, B5: protein expression of p62. Control: control group, Sham: sham-operated group, Model: ICH model group, NCXP (L): low-dose Naochuxue prescription (0.13 g/kg), NCXP (M): medium-dose Naochuxue prescription (0.26 g/kg), NCXP (H): high-dose Naochuxue prescription (0.52 g/kg). The drug was administered by once daily for 10 d. NCXP: Naochuxue prescription; ICH: intracerebral hemorrhage; HMGB1: high mobility group box-1; LC3: microtubule-associated protein 1 light chain 3 beta; p62: sequestosome 1. One-way analysis of variance, and Tukey's multiple comparisons test. Results are presented as mean ± standard deviation. aP>0.05, bP<0.01, fP<0.05, vs Control; cP<0.05, dP<0.01, eP>0.05, vs Model, n = 6 in each group.

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